Orphanin FQ receptor

ABSTRACT

The present invention provides Orphanin FQ receptor nucleic acids and polypeptides and uses thereof. In particular, the present invention provides nucleic acid sequences of differentially expressed splice variants of the Orphanin FQ receptor. The present invention also provides methods of using the Orphanin FQ receptor nucleic acid sequences and ligands for the identification of pharmaceutical agents, the generation of animal models of Orphanin FQ receptor-mediated disease states, and for formulating biological activities. The present invention further provides improved methods of screening potential therapeutics useful in the treatment of a variety of disease states mediated by Orphanin FQ signaling, as well as therapeutics identified using the screening methods.

The present invention claims priority to U.S. Provisional PatentApplication Ser. No. 60/471,642, filed May 19, 2003, which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides Orphanin FQ receptor nucleic acids andpolypeptides and uses thereof. In particular, the present inventionprovides nucleic acid sequences of differentially expressed splicevariants of the Orphanin FQ receptor. The present invention alsoprovides methods of using the Orphanin FQ receptor nucleic acidsequences and ligands for the identification of pharmaceutical agents,the generation of animal models of Orphanin FQ receptor-mediated diseasestates, and for formulating biological activities. The present inventionfurther provides improved methods of screening potential therapeuticsuseful in the treatment of a variety of disease states mediated byOrphanin FQ signaling, as well as therapeutics identified using thescreening methods.

BACKGROUND

The use (and abuse) of opiates, archetypally opium and morphine, havebeen known since antiquity (reviewed in Brownstein, Proc. Natl. Acad.Sci. USA 90:5391 [1993]). Since the nineteenth century, chemicalcharacterization and synthesis of a number of morphine analogues havebeen achieved in an effort to discover a compound with the analgesiceffects of morphine that lacks or is substantially attenuated in itsaddictive potential. These efforts have proven fruitless to date.

The biology behind the reasons why morphine and morphine-like compoundsdisplay both analgesic and addictive properties was first elucidated bythe discovery of endogenous morphine-like compounds termed enkephalins(See e.g., DiChara and North, Trends in Pharmacol. Sci. 13:185 [1992]for review). Accompanying this finding of an endogenous opiate was thebiochemical evidence for a family of related but distinct opiatereceptors, each of which displays a unique pharmacological profile ofresponse to opiate agonists and antagonists (See e.g., McKnight andRees, Neurotransmissions 7:1 [1991] for review). To date, four distinctopiate receptors have been described by their pharmacological profilesand anatomical distribution: these comprise the mu, delta, kappa andsigma receptors.

In 1991, U.S. pharmaceutical companies spent an estimated $7.9 billionon research and development devoted to identifying new therapeuticagents (Pharmaceutical Manufacturer's Association). The magnitude ofthis amount is due, in part, to the fact that the hundreds, if notthousands, of chemical compounds must be tested in order to identify asingle effective therapeutic agent that does not engender unacceptablelevels of undesirable or deleterious side effects. There is anincreasing need for economical methods of testing large numbers ofchemical compounds to quickly identify those compounds that are likelyto be effective in treating disease.

This is of particular importance for psychoactive and psychotropicdrugs, due to their pharmacological importance and their potential togreatly benefit or greatly harm human patients treated with such drugs.At present, few such economical systems exist. Conventional screeningmethods require the use of animal brain slices in binding assays as afirst step. This is suboptimal for a number of reasons, includinginterference in the binding assay by non-specific binding ofheterologous (i.e., non-receptor) cell surface proteins expressed bybrain cells in such slices; differential binding by cells other thanneuronal cells present in the brain slice, such as glial cells or bloodcells; and the possibility that putative drug binding behavior in animalbrain cells will differ from the binding behavior in human brain cellsin subtle but critical ways. For these and other reasons, development ofin vitro screening methods for psychotropic drugs has numerousadvantages and is a major research goal in the pharmaceutical industry.

SUMMARY OF THE INVENTION

The present invention provides Orphanin FQ receptor nucleic acids andpolypeptides and uses thereof. In particular, the present inventionprovides nucleic acid sequences of differentially expressed splicevariants of the Orphanin FQ receptor. The present invention alsoprovides methods of using the Orphanin FQ receptor nucleic acidsequences and ligands for the identification of pharmaceutical agents,the generation of animal models of Orphanin FQ receptor-mediated diseasestates, and for formulating biological activities. The present inventionfurther provides improved methods of screening potential therapeuticsuseful in the treatment of a variety of disease states mediated byOrphanin FQ signaling, as well as therapeutics identified using thescreening methods.

For example, in some embodiments, the present invention providescompositions and methods for altering (e.g., increasing)gastrointestinal function, and colonic transit in particular, useful inthe treatment of colonic motility disorders including, but not limitedto, constipation (e.g., encopresis), colonic inertia, diarrhiadisorders, irritable bowel disorder (IBD), mega colon, colonic pseudoobstruction, and post operative ileus.

In some embodiments, the present in provides ligands that stimulateOFQR. In other embodiments, the present invention provides ligands thatdecrease the function of OFQR.

Some other embodiments provide pharmaceutical compositions comprising:an OFQR ligand (e.g., peptide, peptide mimetic, antibody, smallmolecule, nucleic acid, and the like) as described herein; and/orinstructions for administering the composition to a subject, the subjectcharacterized as having a disease state (e.g., colonic transitdisorder). In some of these embodiments, the subject's colonic transitdisorder is resistant to existing treatments. In preferred embodiments,the instructions for administering the pharmaceutical composition meetUS Food and Drug Administrations (USFDA) rules, regulations, andsuggestions for the provision of therapeutic compounds.

Other advantages, benefits, and preferable embodiments of the presentinvention will be apparent to those skilled in the art.

DESCRIPTION OF THE FIGURES

The following figures form part of the present specification and areincluded to further demonstrate certain aspects and embodiments of thepresent invention. The invention may be better understood by referenceto one or more of these figures in combination with the description ofspecific embodiments presented herein.

FIG. 1 shows the structure of the rat OFQ receptor gene. (A) Restrictionmap and exon location. The restriction sites for Pst I (P), Acc I (A),and Sac I (S) are shown with the relative distances of each exon. (B)mRNA organization. Shaded and clear rectangles with size (bp) representexons of the open reading frame and the untranslated regions,respectively. The ATG translation initiation codon is located in exon 2,and the TGA termination codon is located in exon 6.

FIG. 2 shows the nucleotide (SEQ ID NO:34) and deduced amino acid (SEQID NO:35) sequences of the rat OFQ receptor gene. The five exons(uppercase) are underlined. The nucleotide sequence is numbered on thefar left, relative to the first nucleotide of the translation initiationcodon in exon 2. Amino acids are numbered on the far right and aredesignated with single-letter symbols below each triplet codon. FIG. 2also shows the nucleotide sequence of the 5′ flanking region of the ratOFQ receptor gene. The nucleotide sequence is numbered relative to thefirst nucleotide (dA) of the translation initiation codon (far right). Atandem dinucleotide sequence (GA) at nt 2312–2251 and two (AAAAC)₃ (SEOID NO:43) repeat sequences at nt 1106–1053 are underlined. Potentialbinding sites for RNA polymerase II (TATA) are marked with boxes at nt852–849 and at nt 272–269. Transcription initiation sites are indicatedwith arrows. The exons are in uppercase. The methionine (M) of thetranslation initiation codon is depicted with a single-letter symbolbelow the triplet codon (ATG) in exon 2. The sites of consensus motifsfor transcription factors Ap2 and Sp1 are underlined and labeled.

FIG. 3 shows primer extension (SEQ ID NOS: 41–42) for the identificationof the transcription initiation sites. The transcription initiationsites were identified as the cytosine at nt 750 (A) and the guanine atnt 139 (B) (see asterisks). Lanes A, C, G, and T indicate the size ofthe genomic DNA from dideoxy sequencing using the same primers that wereused for the primer extension experiment. (C) Transcription initiationsites. CAP 1 is the transcription initiation site of mRNAs containingexon 1 by joining nt 501 in exon 1 to nt 33 in exon 2. CAP 2 is thetranscription initiation site of mRNAs deleted for exon 1.

FIG. 4 shows PCR cloning of gene splice variants. The resulting PCRbands obtained from primers P5 and P10 using the RT-PCR products ofeither primer P9 located in exon 1 (A) or P3 contained in intron 1sequence (B) in combination with primer P13, were subcloned into the M13vector and sequenced by the dideoxy chain termination method. (C)Alternative splicing of the rat OFQR gene. OFQR-a (SEQ ID NO:10)contained all six exons, whereas OFQR-e was deleted for exons 3, 4, and5. Two mRNA variants, OFQR-b (SEQ ID NO:12) and -c (SEQ ID NO:14) weredeleted for exon 4 and for exons 3 and 4, respectively. OFQR-d (SEQ IDNO:16) was the same isoform as OFQR-c, except for the 15-bp(GTATGTCATCCTCAG; SEQ ID NO:36) deletion in exon 2 at nt 243–257.OFQR-b′ (SEQ ID NO:17), -c′ (SEQ ID NO:19), -d′ (SEQ ID NO:20) and -e′(SEQ ID NO:21) are mRNA variants deleted for exon 1. Shaded and clearboxes indicate exons for the open reading frames (corresponding to thepolypeptide sequences for variant a (SEQ ID NO:11); variant b (SEQ IDNO:13); variant c (SEQ ID NO:15; variant d (SEQ ID NO:17; and variant e(SEQ ID NO:23) for the untranslated regions, respectively. The asterisksin exons 5 and 6 indicate the positions of early termination in proteincoding regions. The putative open reading frames are (D) Translation ofintron 5 and comparison of the amino acid homology to the publishedsequence. The nucleotide sequence (a; SEQ ID NO:31) of intron 5 isindicated by arrows and is depicted in the single-letter symbols beloweach triplet codon (b; SEQ ID NO:32), and compared to the amino acidsequence reported by Wang et al. (FEBS Lett. 348, 75 [1994]) (c; SEQ IDNO:33).

FIG. 5 shows Expression of alternative splicing in rat tissues. Eitherprimer P9 (A) in exon 1 or P3 (B) in intron 1 in combination with P13 inthe 3′ untranslated region were used in PCR to produce full-lengthcDNAs. The primer P3 containing the 19-mer intron 1 sequence (shown inlowercase in Table 1) was designed to verify only exon 2 expression inmRNA. OFQR-c, -d, -c′ and -d′ could not be distinguished from oneanother due to minimal size differences (i.e., 15 bp). No message couldbe detected from kidney, heart, lung, or muscle. Multiple receptor formscontaining exon 1 were expressed in various tissues (A), whereas thosewithout exon 1 were restricted to brain (B). GAPDH served as an internalcontrol (C).

FIG. 6 shows the nucleic acid sequence of SEQ ID NO: 9.

FIG. 7 shows the nucleic acid sequence of SEQ ID NO: 10.

FIG. 8 shows the amino acid sequence of SEQ ID NO: 11.

FIG. 9 shows the nucleic acid sequence of SEQ ID NO: 12.

FIG. 10 shows the amino acid sequence of SEQ ID NO: 13.

FIG. 11 shows the nucleic acid sequence of SEQ ID NO: 14.

FIG. 12 shows the amino acid sequence of SEQ ID NO: 15.

FIG. 13 shows the nucleic acid sequence of SEQ ID NO: 16.

FIG. 14 shows the amino acid sequence of SEQ ID NO: 17.

FIG. 15 shows the nucleic acid sequence of SEQ ID NO: 18.

FIG. 16 shows the nucleic acid sequence of SEQ ID NO: 19.

FIG. 17 shows the nucleic acid sequence of SEQ ID NO: 20.

FIG. 18 shows the nucleic acid sequence of SEQ ID NO: 21.

FIG. 19 shows the nucleic acid sequence of SEQ ID NO: 22.

FIG. 20 shows the amino acid sequence of SEQ ID NO: 23.

GENERAL DESCRIPTION OF THE INVENTION

The present invention provides Orphanin FQ receptor nucleic acidsequences. Specifically, the present invention provides nucleic acidsequence of differentially expressed splice variants of the Orphanin FQreceptor. The present invention also provides methods of using theOrphanin FQ receptor nucleic acid sequences for the identification ofpharmaceutical agents and the generation of animal models of Orphanin FQreceptor-mediated disease states.

Orphanin FQ (OFQ), also called nociceptin, is a 17-amino acid peptide(Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln;SEQ ID NO:24) isolated from the central nervous system. Research hasshown that OFQ receptors (OFQRs) have the classical seventransmembrane-spanning domain structure of G protein-linked receptorsand high sequence homology to opioid receptors (Bunzow et al., FEBSLett. 347: 284 [1994]). OFQRs bear several similarities to opioidreceptors in that they appear to inhibit adenylate cyclase through aG_(i) protein, they activate inwardly rectifying K⁺ channels, and theyinhibit N-type voltage-operated Ca²⁺ channels. However, OFQRs have avery low affinity for the classical opioid ligands (Darland et al.,Trends Neurosci. 21, 215 [1998]). Similar or perhaps identical receptorsin various species have been reported: in human, opioid receptor-like 1(ORL1, Mollereau et al., FEBS Lett. 341, 331994); in rat, rat opioidreceptor C (ROR-C, Fukuda et al., FEBS Lett. 343:42 [1994]), X-opioidreceptor (XOR, Chen et al., FEBS Lett. 347:279 [1994]), LC132 (Bunzow etal., FEBS Lett. 347:284 [1994]), X-opioid receptor 1 (XOR1, Wang et al.,supra), Hyp 8-1 (Wick et al., Brain Res. Mol. Brain Res. 27:37 [1994]),and C3 (Lachowicz et al., J. Neurochem. 64: 34 [1995]); and in mouse,opioid receptor C (MOR-C, Nishi et al., Biochem. Biophys. Res. Commun.205:1353 [1994]) and κ₃-related opioid receptor (KOR-3, Pan et al.,Regul. Pept. 54, 217 [1994]; Pan et al., Mol. Pharmacol. 47:1180 [1995];Pan et al., Gene 171:255 [1996a]). OFQ binds to these receptors withhigh affinity in a saturable manner (Reinscheid et al., Science 270:792[1995]). Moreover, OFQ inhibits (with an EC₅₀ of about 1 nM)forskolin-induced cAMP accumulation in CHO cells transfected with thereceptor; an effect that is not modified by opioid ligands (Meunier etal., Nature 377:532 [1995]; Reinscheid et al., supra). OFQ was shown toreduce, in a concentration-dependent manner, the electrically inducedileal contraction in guinea pig (Zhang et al., Brain Res. 772:102[1997]), rat (Yazdani et al., Gastroenterology 116:108 [1999]), and pig(Osinski et al., Eur. J. Pharmacol. 365:281 [1999]).Intracerebroventricular OFQ administration was shown to inducehyperalgesia and decrease locomotor activity in mice (Meunier et al.,supra; Reinscheid et al., supra), suggesting a modulatory orneurotransmitter role in the central nervous system, as has beenreported for other central functions (for a review, see Darland et al.,supra). Subsequently, OFQ was shown to have a dual effect on painperception in mice—initial hyperalgesia followed by analgesia (Rossi etal., J. Pharmacol. Exp. Ther. 282:858 [1997]). Although the analgesiceffect can be blocked by classical opioid antagonists, it is notmediated by traditional δ-, κ- and μ-opioid receptors (Rossi et al.,[1997] supra; Rossi et al., Brain Res. 792:327 [1998]; Noda et al., J.Biol. Chem. 273:18047 [1998]).

These observations suggest that OFQR subtypes may exist in the centralnervous system. This hypothesis is supported by receptor binding studiesof cerebral homogenates or CHO cells that express OFQR chimeras (Mathiset al., Biochem. Biophys. Res. Commun. 230:462 [1997]; Pan et al., FEBSLett. 395:207 [1996b]). OFQR mRNA expression has also been demonstratedin peripheral tissues such as rat intestine (Wang et al., supra), piggastrointestinal tract (Osinski et al., 1999), and in human immunesystem cells such as lymphocytes and monocytes (Peluso et al., supra).

Accordingly, in some embodiments, the present invention provides genesencoding OFQ receptors, as well as isoforms (e.g., splice variants)expressed in different tissues. In other embodiments, the presentinvention provides drug screening assays for the identification ofcompounds that effect signaling by the different isoforms of the OFQR(e.g., agonists or antagonists). The present invention is not limited toany one mechanism. Indeed, an understanding of the mechanism is notnecessary to practice the present invention. Nonetheless, it iscontemplated that therapeutic agents are targeted to specific receptorisoforms, and can thus provide specific therapies with decreased sideeffects.

The present invention further provides therapeutic agents that targetOFRQ. Experiments conducted during the course of development of thepresent invention demonstrated that Orphanin FQ peptide increasescolonic motility. Accordingly, in some embodiments, the presentinvention provides methods of treating disorders of colonic motility(e.g., post-operative ileitis) by administering agonists or antagonistsof OFQR (e.g., including, but not limited to, Orphanin mimetics andother small molecules).

Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein the term “disease” refers to a deviation from thecondition regarded as normal or average for members of a species, andwhich is detrimental to an affected individual under conditions that arenot inimical to the majority of individuals of that species (e.g.,diarrhea, nausea, fever, pain, and inflammation etc).

As used herein the term “colonic transit disorder” refers to a deviationfrom the condition regarded as normal or average for members of aspecies, and which is detrimental to an affected individual underconditions that are not inimical to the majority of individuals of thatspecies involving the transit of fecal material through the intestines,and in particular through the colon (i.e., ascending, transverse,descending, and sigmoid), rectum, anal canal, and anus.

As used herein, the term “therapeutic agent,” refers to compositionsthat decrease the symptoms of a disease in a host. Such agents mayadditionally comprise pharmaceutically acceptable compounds (e.g.,adjutants, excipients, stabilizers, diluents, and the like). In someembodiments, the therapeutic agents are administered in the form oftopical emulsions, injectable compositions, ingestible solutions, andthe like. When the route is topical, the form may be, for example, aspray (e.g., a nasal spray).

The terms “pharmaceutically acceptable” or “pharmacologicallyacceptable,” as used herein, refer to compositions that do notsubstantially produce adverse allergic or immunological reactions whenadministered to a host (e.g., an animal or a human). As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, wetting agents (e.g., sodium laurylsulfate), isotonic and absorption delaying agents, disintrigrants (e.g.,potato starch or sodium starch glycolate), and the like.

As used herein, the term “systemically active drugs” is used broadly toindicate a substance or composition that will produce a pharmacologicalresponse at a site remote from the point of application or entry into asubject.

As used herein, the term “agonist” refers to a compound (e.g., a drug)that has affinity for the cellular receptor (e.g., OFQR) of another drugor natural substance (e.g., Orphanin FQ) and that produces aphysiological effect.

As used herein, the term “antagonist” refers to a compound (e.g., adrug) that binds to a cellular receptor (e.g., OFQR) for a hormone,neurotransmitter (e.g., Orphanin FQ), or another drug blocking theaction of that substance without producing any physiologic effectitself.

As used herein, the term “mimetic” refers to a small molecule compoundthat mimics the binding of a ligand to its target.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of a polypeptideor precursor (e.g., OFQR). The polypeptide can be encoded by a fulllength coding sequence or by any portion of the coding sequence so longas the desired activity or functional properties (e.g., ligand binding,signal transduction, etc.) of the full-length or fragment are retained.The term also encompasses the coding region of a structural gene and theincluding sequences located adjacent to the coding region on both the 5′and 3′ ends for a distance of about 1 kb on either end such that thegene corresponds to the length of the full-length mRNA. The sequencesthat are located 5′ of the coding region and which are present on themRNA are referred to as 5′ untranslated sequences. The sequences thatare located 3′ or downstream of the coding region and that are presenton the mRNA are referred to as 3′ untranslated sequences. The term“gene” encompasses both cDNA and genomic forms of a gene. A genomic formor clone of a gene contains the coding region interrupted withnon-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are segments of a gene that aretranscribed into nuclear RNA (hnRNA); introns may contain regulatoryelements such as enhancers. Introns are removed or “spliced out” fromthe nuclear or primary transcript; introns therefore are absent in themessenger RNA (mRNA) transcript. The mRNA functions during translationto specify the sequence or order of amino acids in a nascentpolypeptide.

In particular, the term “OFQR gene” refers to the full-length OFQRnucleotide sequence (e.g., contained in SEQ ID NO:9). Furthermore, theterms “OFQR nucleotide sequence” or “OFQR polynucleotide sequence”encompasses DNA, cDNA, and RNA (e.g., mRNA) sequences. It is alsointended that the term encompass splice variants of OFQR (e.g.,including but not limited to SEQ ID NOs: 10, 12, 14, 16, 18, 19, 20, and21).

As used herein, the term “OFQR polypeptide” refers to polypeptidesencoded by an OFQR gene. The term is intended to encompass variants ofOFQR (e.g., splice variants of SEQ ID NOs: 11, 13, 15, 17, and 23) aswell as mutants, homologs, and orthologs thereof.

As used herein, the terms “interact,” “interaction,” and “level ofinteraction” refer to a physical interaction (e.g., binding) between apolypeptide (e.g., an OFQR polypeptide) and a molecule (e.g., a ligandor a test compound). The level of interaction can be determined, forexample, by using one of the binding assays described in Section IVbelow.

As used herein, the term “capable of binding” as in “capable of bindingto orphanin FQ” refers to a polypeptide (e.g., an OFQR polypeptide) thatis able to bind to a molecule (e.g., a ligand including but not limitedto, orphanin FQ). Binding can be measured using any suitable assay,including but not limited to, those disclosed herein (See e.g., SectionIV below).

As used herein the term “OFQR signaling activity” refers to one of thesignaling activities mediated by the binding of orphanin FQ to OFQR(e.g., including but not limited to, one of the activities disclosedherein). Signaling can be measured using any suitable assay, includingbut not limited to, the reporter gene assay described in Section IVbelow. As used herein, the term “altering OFQR signaling activity” as in“the ability of a test compound to alter OFQR signaling activity” refersto an altered level of signaling (e.g., higher or lower) relative to thelevel of signaling in the absence of a test compound.

Where amino acid sequence is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, amino acid sequenceand like terms, such as polypeptide or protein are not meant to limitthe amino acid sequence to the complete, native amino acid sequenceassociated with the recited protein molecule.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequencesthat are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region may contain sequencesthat direct the termination of transcription, post-transcriptionalcleavage and polyadenylation.

The term “wild-type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the terms“modified”, “mutant”, and “variant” refer to a gene or gene product thatdisplays modifications in sequence and or functional properties (i.e.,altered characteristics) when compared to the wild-type gene or geneproduct. It is noted that naturally-occurring mutants can be isolated;these are identified by the fact that they have altered characteristicswhen compared to the wild-type gene or gene product.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides or polynucleotidesin a manner such that the 5′ phosphate of one mononucleotide pentosering is attached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage. Therefore, an end of an oligonucleotides orpolynucleotide, referred to as the “5′ end” if its 5′ phosphate is notlinked to the 3′ oxygen of a mononucleotide pentose ring and as the “3′end” if its 3′ oxygen is not linked to a 5′ phosphate of a subsequentmononucleotide pentose ring. As used herein, a nucleic acid sequence,even if internal to a larger oligonucleotide or polynucleotide, also maybe said to have 5′ and 3′ ends. In either a linear or circular DNAmolecule, discrete elements are referred to as being “upstream” or 5′ ofthe “downstream” or 3′ elements. This terminology reflects the fact thattranscription proceeds in a 5′ to 3′ fashion along the DNA strand. Thepromoter and enhancer elements that direct transcription of a linkedgene are generally located 5′ or upstream of the coding region. However,enhancer elements can exert their effect even when located 3′ of thepromoter element and the coding region. Transcription termination andpolyadenylation signals are located 3′ or downstream of the codingregion.

As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a gene” and “polynucleotide having a nucleotidesequence encoding a gene,” means a nucleic acid sequence comprising thecoding region of a gene or, in other words, the nucleic acid sequencethat encodes a gene product. The coding region may be present in eithera cDNA, genomic DNA, or RNA form. When present in a DNA form, theoligonucleotide or polynucleotide may be single-stranded (i.e., thesense strand) or double-stranded. Suitable control elements such asenhancers/promoters, splice junctions, polyadenylation signals, etc. maybe placed in close proximity to the coding region of the gene if neededto permit proper initiation of transcription and/or correct processingof the primary RNA transcript. Alternatively, the coding region utilizedin the expression vectors of the present invention may containendogenous enhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

As used herein, the term “regulatory element” refers to a geneticelement that controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element thatfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements include splicing signals,polyadenylation signals, termination signals, etc.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, for the sequence“A-G-T,” is complementary to the sequence “T-C-A.” Complementarity maybe “partial,” in which only some of the nucleic acids' bases are matchedaccording to the base pairing rules. Or, there may be “complete” or“total” complementarity between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods that depend upon binding between nucleicacids.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid and is referred to using the functional term “substantiallyhomologous.” The term “inhibition of binding,” when used in reference tonucleic acid binding, refers to inhibition of binding caused bycompetition of homologous sequences for binding to a target sequence.The inhibition of hybridization of the completely complementary sequenceto the target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially homologous sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely homologous to a target under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target that lacks even a partial degreeof complementarity (e.g., less than about 30% identity); in the absenceof non-specific binding the probe will not hybridize to the secondnon-complementary target.

The art knows well that numerous equivalent conditions may be employedto comprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)are considered and the hybridization solution may be varied to generateconditions of low stringency hybridization different from, butequivalent to, the above listed conditions. In addition, the art knowsconditions that promote hybridization under conditions of highstringency (e.g., increasing the temperature of the hybridization and/orwash steps, the use of formamide in the hybridization solution, etc.).

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described above.

A gene may produce multiple RNA species that are generated bydifferential splicing of the primary RNA transcript. cDNAs that aresplice variants of the same gene (e.g., the splice variants of OFQRrepresented by SEQ ID NOs: 10, 12, 14, 16, 18, 19, 20, and 21) willcontain regions of sequence identity or complete homology (representingthe presence of the same exon or portion of the same exon on both cDNAs)and regions of complete non-identity (for example, representing thepresence of exon “A” on cDNA 1 wherein cDNA 2 contains exon “B”instead). Because the two cDNAs contain regions of sequence identitythey will both hybridize to a probe derived from the entire gene orportions of the gene containing sequences found on both cDNAs; the twosplice variants are therefore substantially homologous to such a probeand to each other.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(i.e., it is the complement of) the single-stranded nucleic acidsequence under conditions of low stringency as described above.

As used herein, the term “competes for binding” is used in reference toa first polypeptide with an activity which binds to the same substrateas does a second polypeptide with an activity, where the secondpolypeptide is a variant of the first polypeptide or a related ordissimilar polypeptide. The efficiency (e.g., kinetics orthermodynamics) of binding by the first polypeptide may be the same asor greater than or less than the efficiency substrate binding by thesecond polypeptide. For example, the equilibrium binding constant(K_(D)) for binding to the substrate may be different for the twopolypeptides. The term “K_(M)” as used herein refers to theMichaelis-Menton constant for an enzyme and is defined as theconcentration of the specific substrate at which a given enzyme yieldsone-half its maximum velocity in an enzyme catalyzed reaction.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization [1985]). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Those skilled in the art will recognizethat “stringency” conditions may be altered by varying the parametersjust described either individually or in concert. With “high stringency”conditions, nucleic acid base pairing will occur only between nucleicacid fragments that have a high frequency of complementary basesequences (e.g., hybridization under “high stringency” conditions mayoccur between homologs with about 85–100% identity, preferably about70–100% identity). With medium stringency conditions, nucleic acid basepairing will occur between nucleic acids with an intermediate frequencyof complementary base sequences (e.g., hybridization under “mediumstringency” conditions may occur between homologs with about 50–70%identity). Thus, conditions of “weak” or “low” stringency are oftenrequired with nucleic acids that are derived from organisms that aregenetically diverse, as the frequency of complementary sequences isusually less.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42 C in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42 C when aprobe of about 500 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42 C in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42 C when aprobe of about 500 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42 C in a solution consisting of 5×SSPE (43.8 g/l NaCl,6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH),0.1% SDS, 5× Denhardt's reagent [50× Denhardt's contains per 500 ml: 5 gFicoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 g/mldenatured salmon sperm DNA followed by washing in a solution comprising5×SSPE, 0.1% SDS at 42 C when a probe of about 500 nucleotides in lengthis employed.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence,” “sequenceidentity,” “percentage of sequence identity,” and “substantialidentity.” A “reference sequence” is a defined sequence used as a basisfor a sequence comparison; a reference sequence may be a subset of alarger sequence, for example, as a segment of a full-length cDNAsequence given in a sequence listing or may comprise a complete genesequence. Generally, a reference sequence is at least 20 nucleotides inlength, frequently at least 25 nucleotides in length, and often at least50 nucleotides in length. Since two polynucleotides may each (1)comprise a sequence (i.e., a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2) mayfurther comprise a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window,” as usedherein, refers to a conceptual segment of at least 20 contiguousnucleotide positions wherein a polynucleotide sequence may be comparedto a reference sequence of at least 20 contiguous nucleotides andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman [Smithand Waterman, Adv. Appl. Math. 2:482 (1981)] by the homology alignmentalgorithm of Needleman and Wunsch [Needleman and Wunsch, J. Mol. Biol.48:443 (1970)], by the search for similarity method of Pearson andLipman [Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444(1988)], by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.),or by inspection, and the best alignment (i.e., resulting in the highestpercentage of homology over the comparison window) generated by thevarious methods is selected. The term “sequence identity” means that twopolynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity.

As applied to polynucleotides, the term “substantial identity” denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 85 percent sequence identity,preferably at least 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 nucleotide positions, frequentlyover a window of at least 25–50 nucleotides, wherein the percentage ofsequence identity is calculated by comparing the reference sequence tothe polynucleotide sequence which may include deletions or additionswhich total 20 percent or less of the reference sequence over the windowof comparison. The reference sequence may be a subset of a largersequence, for example, as a splice variant of the full-length sequencesof the compositions claimed in the present invention (e.g., OFQR).

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions that are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Q replicase, MDV-1 RNA is the specific template for thereplicase (D. L. Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038[1972]). Other nucleic acid will not be replicated by this amplificationenzyme. Similarly, in the case of T7 RNA polymerase, this amplificationenzyme has a stringent specificity for its own promoters (M. Chamberlinet al., Nature 228:227 [1970]). In the case of T4 DNA ligase, the enzymewill not ligate the two oligonucleotides or polynucleotides, where thereis a mismatch between the oligonucleotide or polynucleotide substrateand the template at the ligation junction (D. Y. Wu and R. B. Wallace,Genomics 4:560 [1989]). Finally, Taq and Pfu polymerases, by virtue oftheir ability to function at high temperature, are found to display highspecificity for the sequences bounded and thus defined by the primers;the high temperature results in thermodynamic conditions that favorprimer hybridization with the target sequences and not hybridizationwith non-target sequences (H. A. Erlich (ed.), PCR Technology, StocktonPress [1989]).

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids that may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of “target”(defined below). In contrast, “background template” is used in referenceto nucleic acid other than sample template that may or may not bepresent in a sample. Background template is most often inadvertent. Itmay be the result of carryover, or it may be due to the presence ofnucleic acid contaminants sought to be purified away from the sample.For example, nucleic acids from organisms other than those to bedetected may be present as background in a test sample.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification and isolation of particulargene sequences. It is contemplated that any probe used in the presentinvention will be labeled with any “reporter molecule,” so that isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the present invention be limited to any particular detection systemor label.

As used herein, the term “target,” when used in reference to thepolymerase chain reaction, refers to the region of nucleic acid boundedby the primers used for polymerase chain reaction. Thus, the “target” issought to be sorted out from other nucleic acid sequences. A “segment”is defined as a region of nucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of K. B. Mullis (See e.g., U.S. Pat. Nos. 4,683,195,4,683,202, and 4,965,188, hereby incorporated by reference), whichdescribe a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification. This process for amplifying the target sequence consistsof introducing a large excess of two oligonucleotide primers to the DNAmixture containing the desired target sequence, followed by a precisesequence of thermal cycling in the presence of a DNA polymerase. The twoprimers are complementary to their respective strands of the doublestranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing, and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one “cycle”; there can be numerous “cycles”) toobtain a high concentration of an amplified segment of the desiredtarget sequence. The length of the amplified segment of the desiredtarget sequence is determined by the relative positions of the primerswith respect to each other, and therefore, this length is a controllableparameter. By virtue of the repeating aspect of the process, the methodis referred to as the “polymerase chain reaction” (hereinafter “PCR”).Because the desired amplified segments of the target sequence become thepredominant sequences (in terms of concentration) in the mixture, theyare said to be “PCR amplified.”

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTPor dATP, into the amplified segment). In addition to genomic DNA, anyoligonucleotide or polynucleotide sequence can be amplified with theappropriate set of primer molecules. In particular, the amplifiedsegments created by the PCR process itself are, themselves, efficienttemplates for subsequent PCR amplifications.

As used herein, the terms “PCR product,” “PCR fragment,” and“amplification product” refer to the resultant mixture of compoundsafter two or more cycles of the PCR steps of denaturation, annealing andextension are complete. These terms encompass the case where there hasbeen amplification of one or more segments of one or more targetsequences.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template, and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the term “recombinant DNA molecule” as used hereinrefers to a DNA molecule that is comprised of segments of DNA joinedtogether by means of molecular biological techniques.

As used herein, the term “antisense” is used in reference to RNAsequences that are complementary to a specific RNA sequence (e.g.,mRNA). The term “antisense strand” is used in reference to a nucleicacid strand that is complementary to the “sense” strand. The designation(−) (i.e., “negative”) is sometimes used in reference to the antisensestrand, with the designation (+) sometimes used in reference to thesense (i.e., “positive”) strand.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant nucleic acid with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is present in a form or settingthat is different from that in which it is found in nature. In contrast,non-isolated nucleic acids are nucleic acids such as DNA and RNA foundin the state they exist in nature. For example, a given DNA sequence(e.g., a gene) is found on the host cell chromosome in proximity toneighboring genes; RNA sequences, such as a specific mRNA sequenceencoding a specific protein, are found in the cell as a mixture withnumerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acids encoding OFQR include, by way of example, suchnucleic acid in cells ordinarily expressing OFQR where the nucleic acidis in a chromosomal location different from that of natural cells, or isotherwise flanked by a different nucleic acid sequence than that foundin nature. The isolated nucleic acid, oligonucleotide, or polynucleotidemay be present in single-stranded or double-stranded form. When anisolated nucleic acid, oligonucleotide or polynucleotide is to beutilized to express a protein, the oligonucleotide or polynucleotidewill contain at a minimum the sense or coding strand (i.e., theoligonucleotide or polynucleotide may single-stranded), but may containboth the sense and anti-sense strands (i.e., the oligonucleotide orpolynucleotide may be double-stranded).

As used herein the term “portion” when in reference to a nucleotidesequence (as in “a portion of a given nucleotide sequence”) refers tofragments of that sequence. The fragments may range in size from fournucleotides to the entire nucleotide sequence minus one nucleotide (10nucleotides, 20, 30, 40, 50, 100, 200, etc.).

As used herein the term “coding region” when used in reference tostructural gene refers to the nucleotide sequences that encode the aminoacids found in the nascent polypeptide as a result of translation of amRNA molecule. The coding region is bounded, in eukaryotes, on the 5′side by the nucleotide triplet “ATG” that encodes the initiatormethionine and on the 3′ side by one of the three triplets that specifystop codons (i.e., TAA, TAG, TGA).

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample. For example, OFQR antibodies are purifiedby removal of contaminating non-immunoglobulin proteins; they are alsopurified by the removal of immunoglobulin that does not bind OFQR orspecific splice variants of OFQR. The removal of non-immunoglobulinproteins and/or the removal of immunoglobulins that do not bind OFQR orsplice variants thereof results in an increase in the percent ofOFQR-reactive immunoglobulins in the sample.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule that is expressed from a recombinantDNA molecule.

The term “native protein” as used herein to indicate that a protein doesnot contain amino acid residues encoded by vector sequences; that is thenative protein contains only those amino acids found in the protein asit occurs in nature. A native protein may be produced by recombinantmeans or may be isolated from a naturally occurring source.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four consecutive amino acid residues tothe entire amino acid sequence minus one amino acid.

The term “Southern blot,” refers to the analysis of DNA on agarose oracrylamide gels to fractionate the DNA according to size followed bytransfer of the DNA from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized DNA is then probedwith a labeled probe to detect DNA species complementary to the probeused. The DNA may be cleaved with restriction enzymes prior toelectrophoresis. Following electrophoresis, the DNA may be partiallydepurinated and denatured prior to or during transfer to the solidsupport. Southern blots are a standard tool of molecular biologists (J.Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, NY, pp 9.31–9.58 [1989]).

The term “Western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on acrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose or a nylon membrane. The immobilizedproteins are then exposed to antibodies with reactivity against anantigen of interest. The binding of the antibodies may be detected byvarious methods, including the use of labeled antibodies.

The term “antigenic determinant” as used herein refers to that portionof an antigen that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies that bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the “immunogen” used to elicitthe immune response) for binding to an antibody.

The term “transgene” as used herein refers to a foreign gene that isplaced into an organism by introducing the foreign gene into newlyfertilized eggs or early embryos. The term “foreign gene” refers to anynucleic acid (e.g., gene sequence) that is introduced into the genome ofan animal by experimental manipulations and may include gene sequencesfound in that animal so long as the introduced gene does not reside inthe same location as does the naturally-occurring gene.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.”

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

As used herein, the term host cell refers to any eukaryotic orprokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells,mammalian cells, avian cells, amphibian cells, plant cells, fish cells,and insect cells), whether located in vitro or in vivo. For example,host cells may be located in a transgenic animal.

The terms “overexpression” and “overexpressing” and grammaticalequivalents, are used in reference to levels of mRNA to indicate a levelof expression approximately 3-fold higher than that typically observedin a given tissue in a control or non-transgenic animal. Levels of mRNAare measured using any of a number of techniques known to those skilledin the art including, but not limited to Northern blot analysis.Appropriate controls are included on the Northern blot to control fordifferences in the amount of RNA loaded from each tissue analyzed (e.g.,the amount of 28S rRNA, an abundant RNA transcript present atessentially the same amount in all tissues, present in each sample canbe used as a means of normalizing or standardizing the RAD50mRNA-specific signal observed on Northern blots). The amount of mRNApresent in the band corresponding in size to the RNA of interest (e.g.,OFQR splice variants) is quantified; other minor species of RNA thathybridize to the probe are not considered in the quantification of theexpression of the transgenic mRNA.

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transfectant” refers to cells that have taken up foreign DNAbut have failed to integrate this DNA.

The term “calcium phosphate co-precipitation” refers to a technique forthe introduction of nucleic acids into a cell. The uptake of nucleicacids by cells is enhanced when the nucleic acid is presented as acalcium phosphate-nucleic acid co-precipitate. The original technique ofGraham and van der Eb (Graham and van der Eb, Virol., 52:456 [1973]),has been modified by several groups to optimize conditions forparticular types of cells. The art is well aware of these numerousmodifications.

The term “test compound” refers to any chemical entity, pharmaceutical,drug, and the like that are tested in an assay (e.g., a drug screeningassay) for any desired activity (e.g., including but not limited to, theability to treat or prevent a disease, illness, sickness, or disorder ofbodily function, or otherwise alter the physiological or cellular statusof a sample). Test compounds comprise both known and potentialtherapeutic compounds. A test compound can be determined to betherapeutic by screening using the screening methods of the presentinvention. A “known therapeutic compound” refers to a therapeuticcompound that has been shown (e.g., through animal trials or priorexperience with administration to humans) to be effective in suchtreatment or prevention.

The term “sample” as used herein is used in its broadest sense. A samplesuspected of containing a human chromosome or sequences associated witha human chromosome may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern blot analysis), RNA (insolution or bound to a solid support such as for Northern blotanalysis), cDNA (in solution or bound to a solid support) and the like.A sample suspected of containing a protein may comprise a cell, aportion of a tissue, an extract containing one or more proteins and thelike.

As used herein, the term “response,” when used in reference to an assay,refers to the generation of a detectable signal (e.g., accumulation ofreporter protein, increase in ion concentration, accumulation of adetectable chemical product).

As used herein, the term “reporter gene” refers to a gene encoding aprotein that may be assayed. Examples of reporter genes include, but arenot limited to, luciferase (See, e.g., deWet et al., Mol. Cell. Biol.7:725 [1987] and U.S. Pat. Nos. 6,074,859; 5,976,796; 5,674,713; and5,618,682; all of which are incorporated herein by reference), greenfluorescent protein (e.g., GenBank Accession Number U43284; a number ofGFP variants are commercially available from CLONTECH Laboratories, PaloAlto, Calif.), chloramphenicol acetyltransferase, galactosidase,alkaline phosphatase, and horse radish peroxidase.

As used herein, the term “purified” refers to molecules, either nucleicor amino acid sequences, which are removed from their naturalenvironment, isolated or separated. An “isolated nucleic acid sequence”is therefore a purified nucleic acid sequence. “Substantially purified”molecules are at least 60% free, preferably at least 75% free, and morepreferably at least 90% free from other components with which they arenaturally associated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides OFQR nucleic acids. In some embodiments,the present invention provides nucleic acids (e.g., mRNA) encoding novelsplice variants of OFQR. In some embodiments, the present inventionprovides methods of utilizing the splice variants for the development ofspecific receptor agonists and antagonists. In still furtherembodiments, the present invention provides transgenic animals useful asanimal models of disease states involving altered OFQR receptors as wellas providing system for the study of complex neurological responses.

I. OFQR Nucleic Acids

In some embodiments, the present invention comprises OFQR nucleic acids.The present invention is not limited to any one particular OFQR nucleicacid. In some embodiments, the present invention comprises genomic(e.g., DNA sequences) encoding an OFQR. In other embodiments, thepresent invention comprises expressed (e.g., mRNA) sequences encodingspliced OFQR encoding sequences. In still further embodiments, thepresent invention comprises mutants, variants, homologs, and orthologsof the disclosed sequences.

A. Isolation and Characterization of Rat OFQR

In some embodiments, the present invention provides rat OFQR nucleicacid sequences. For example, the present invention provides the rat OFQRnucleic acid sequence of SEQ ID NO:9. Examples 1 and 2 describe theisolation and characterization of the rat OFQR gene. The rat OFQR geneexceeds 10 kb in length and contains six exons that are interrupted byfive introns (FIG. 2). Two major transcription initiation sites wereidentified: one in the 5′ flanking region and the other in intron 1.

In other embodiments, the present invention provides mRNA splicevariants of the rat OFQR gene (e.g., SEQ ID NOs: 10, 12, 14, 16, 18, 19,20, and 21). Primer extension analysis was used to identify splicevariants of the OFQR gene (See Examples 2 and 3). The OFQR gene wasfound to be alternatively spliced to yield multiple RNAs. Splicevariants were PCR cloned and sequenced (Example 3). The sequencingresults revealed that rat OFQR expressed at least nine splice variantsdeleted for exon 1, or exons 3 to 5 (See FIG. 5C).

As described above, the varying effects of Orphanin FQ in differenttissues suggested that multiple OFQR subtypes existed in differenttissues. Example 4 describes the detection of differential tissueexpression of the splice variants. As shown in FIG. 5A, PCR products ofOFQR variants were not detected in liver, kidney, heart, lung, spleen,or skeletal muscle. Two forms were expressed in testes. These forms, inaddition to one additional variant, were expressed in pylorus, ileum,and proximal colon. Both forms, OFQR-b and -c (or -d) were expressed inbrain and antrum. OFQR-c (or -d) was expressed in esophagus, fundus,corpus, duodenum, jejunum, and descending colon. As shown in FIG. 5B,the OFQR-c′ or -d′ splice variants were expressed only in brain. Thesedata suggest that unique regions in the 5′ flanking region and in intron1 of the OFQR gene contribute to the regulation of its expression indifferent tissues.

B. Variants of OFQR

In other embodiments of the present invention, variants of the disclosedOFQR sequences are provided. In preferred embodiments, variants resultfrom mutation, (i.e., a change in the nucleic acid sequence) andgenerally produce altered mRNAs or polypeptides whose structure orfunction may or may not be altered. Any given gene may have none, one,or many variant forms. Common mutational changes that give rise tovariants are generally ascribed to deletions, additions or substitutionsof nucleic acids. Each of these types of changes may occur alone, or incombination with the others, and at the rate of one or more times in agiven sequence.

It is contemplated that it is possible to modify the structure of apolypeptide having a function (e.g., OFQR) for such purposes asincreasing or decreasing binding affinity of the OFQR for a ligand(e.g., Orphanin FQ). Such modified peptides are considered functionalequivalents of peptides having an activity of OFQR as defined herein. Amodified peptide can be produced in which the nucleotide sequenceencoding the polypeptide has been altered, such as by substitution,deletion, or addition. In other words, construct “X” can be evaluated inorder to determine whether it is a member of the genus of modified orvariant OFQRs of the present invention as defined functionally, ratherthan structurally. In preferred embodiments, the activity of variant ormutant OFQR (e.g., ligand binding) is evaluated by a previouslydescribed method (See e.g., Meng et al., Mol. Pharmacol. 53:772 [1998];Wnendt et al., Mol. Pharmacol. 56:334 [1999]). Accordingly, in someembodiments, the present invention provides nucleic acids encoding anOFQR polypeptide that binds Orphanin FQ.

In some embodiments, OFQR nucleic acid sequences are modified to containa portion of the human OFQR cDNA sequence (Gen Bank Accession NumberNM_(—)000913.1; SEQ ID NO:22). In some embodiments, domains (e.g., thosecorresponding to Exons 1, 2, 3, 4 5, or 6 of the rat nucleic acid) aresubstituted with the corresponding human domains. Aligning nucleic acidsequences from various species in order to determine conserved regionsor domains can identify suitable domains of the human nucleic acid. Anysuitable software may be used for sequence alignment, including but notlimited to, those disclosed herein.

Moreover, as described above, variant forms of OFQR are alsocontemplated as being equivalent to those peptides and DNA moleculesthat are set forth in more detail herein. For example, it iscontemplated that isolated replacement of a leucine with an isoleucineor valine, an aspartate with a glutamate, a threonine with a serine, ora similar replacement of an amino acid with a structurally related aminoacid (i.e., conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Accordingly, someembodiments of the present invention provide variants of OFQR disclosedherein containing conservative replacements. Conservative replacementsare those that take place within a family of amino acids that arerelated in their side chains. Genetically encoded amino acids can bedivided into four families: (1) acidic (aspartate, glutamate); (2) basic(lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); and (4)uncharged polar (glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1) acidic(aspartate, glutamate); (2) basic (lysine, arginine, histidine), (3)aliphatic (glycine, alanine, valine, leucine, isoleucine, serine,threonine), with serine and threonine optionally be grouped separatelyas aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (e.g., Stryer ed.,Biochemistry, pg. 17–21, 2nd ed, WH Freeman and Co., 1981). Whether achange in the amino acid sequence of a peptide results in a functionalhomolog can be readily determined by assessing the ability of thevariant peptide to function in a fashion similar to the wild-typeprotein. Peptides having more than one replacement can readily be testedin the same manner.

More rarely, a variant includes “nonconservative” changes (e.g.,replacement of a glycine with a tryptophan). Analogous minor variationscan also include amino acid deletions or insertions, or both. Guidancein determining which amino acid residues can be substituted, inserted,or deleted without abolishing biological activity can be found usingcomputer programs (e.g., LASERGENE software, DNASTAR Inc., Madison,Wis.).

As described in more detail below, variants may be produced by methodssuch as directed evolution or other techniques for producingcombinatorial libraries of variants, described in more detail below. Instill other embodiments of the present invention, the nucleotidesequences of the present invention may be engineered in order to alter aOFQR coding sequence including, but not limited to, alterations thatmodify the cloning, processing, localization, secretion, and/orexpression of the gene product. For example, mutations may be introducedusing techniques that are well known in the art (e.g., site-directedmutagenesis to insert new restriction sites, alter glycosylationpatterns, or change codon preference, etc.).

II. OFQR Polypeptides

In some embodiments, the present invention provides OFQR polynucleotidesequences that encode OFQR polypeptide sequences. OFQR polypeptides(e.g., SEQ ID NOs:11, 13, 15, 17 and 23) are described in FIGS. 2, 8,10, 12, 14, and 20. Other embodiments of the present invention providefragments, fusion proteins or functional equivalents of these OFQRproteins. In still other embodiment of the present invention, nucleicacid sequences corresponding to these various OFQR homologs and mutantsmay be used to generate recombinant DNA molecules that direct theexpression of the OFQR homologs and mutants in appropriate host cells.In some embodiments of the present invention, the polypeptide may be anaturally purified product, in other embodiments it may be a product ofchemical synthetic procedures, and in still other embodiments it may beproduced by recombinant techniques using a prokaryotic or eukaryotichost cells (e.g., by bacterial, yeast, higher plant, insect andmammalian cells in culture). In some embodiments, depending upon thehost employed in a recombinant production procedure, the polypeptide ofthe present invention may be glycosylated or may be non-glycosylated. Inother embodiments, the polypeptides of the invention may also include aninitial methionine amino acid residue.

In one embodiment of the present invention, due to the inherentdegeneracy of the genetic code, DNA sequences other than thepolynucleotide sequences of SEQ ID NOs: 9, 10, 12, 14, 16, 18, 19, 20and 21 which encode substantially the same or a functionally equivalentamino acid sequence, may be used to clone and express OFQR variants. Ingeneral, such polynucleotide sequences hybridize to SEQ ID NO: 9, 10,12, 14, 16, 18, 19, 20 and 21 under conditions of high to mediumstringency as described above. As will be understood by those of skillin the art, it may be advantageous to produce OFQR-encoding nucleotidesequences possessing non-naturally occurring codons. Therefore, in somepreferred embodiments, codons preferred by a particular prokaryotic oreukaryotic host (Murray et al., Nucl. Acids Res., 17 [1989]) areselected, for example, to increase the rate of OFQR expression or toproduce recombinant RNA transcripts having desirable properties, such asa longer half-life, than transcripts produced from naturally occurringsequences.

A. Vectors for Production of OFQR

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. In some embodiments of the presentinvention, vectors include, but are not limited to, chromosomal,nonchromosomal and synthetic DNA sequences (e.g., derivatives of SV40,bacterial plasmids, phage DNA; baculovirus, yeast plasmids, vectorsderived from combinations of plasmids and phage DNA, and viral DNA suchas vaccinia, adenovirus, fowl pox virus, and pseudorabies). It iscontemplated that any vector may be used as long as it is replicable andviable in the host.

In particular, some embodiments of the present invention providerecombinant constructs comprising one or more of the sequences asbroadly described above (e.g., SEQ ID NO: 9, 10, 12, 14, 16, 18, 19, 20and 21). In some embodiments of the present invention, the constructscomprise a vector, such as a plasmid or viral vector, into which asequence of the invention has been inserted, in a forward or reverseorientation. In still other embodiments, the heterologous structuralsequence (e.g., SEQ ID NOs: 9, 10, 12, 14, 16, 18, 19, 20 and 21) isassembled in appropriate phase with translation initiation andtermination sequences. In preferred embodiments of the presentinvention, the appropriate DNA sequence is inserted into the vectorusing any of a variety of procedures. In general, the DNA sequence isinserted into an appropriate restriction endonuclease site(s) byprocedures known in the art.

Large numbers of suitable vectors are known to those of skill in theart, and are commercially available. Such vectors include, but are notlimited to, the following vectors: 1) Bacterial—pQE70, pQE60, pQE-9(Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia); and 2) Eukaryotic—pWLNEO, pSV2CAT, pOG44,PXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). Any otherplasmid or vector may be used as long as they are replicable and viablein the host. In some preferred embodiments of the present invention,mammalian expression vectors comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation sites, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking non-transcribedsequences. In other embodiments, DNA sequences derived from the SV40splice, and polyadenylation sites may be used to provide the requirednon-transcribed genetic elements.

In certain embodiments of the present invention, the DNA sequence in theexpression vector is operatively linked to an appropriate expressioncontrol sequence(s) (promoter) to direct mRNA synthesis. Promotersuseful in the present invention include, but are not limited to, the LTRor SV40 promoter, the E. coli lac or trp, the phage lambda P_(L) andP_(R), T3 and T7 promoters, and the cytomegalovirus (CMV) immediateearly, herpes simplex virus (HSV) thymidine kinase, and mousemetallothionein-I promoters and other promoters known to controlexpression of gene in prokaryotic or eukaryotic cells or their viruses.In other embodiments of the present invention, recombinant expressionvectors include origins of replication and selectable markers permittingtransformation of the host cell (e.g., dihydrofolate reductase orneomycin resistance for eukaryotic cell culture, or tetracycline orampicillin resistance in E. coli).

In some embodiments of the present invention, transcription of the DNAencoding one of the polypeptides of the present invention by highereukaryotes is increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp that act on a promoter to increase its transcription.Enhancers useful in the present invention include, but are not limitedto, the SV40 enhancer on the late side of the replication origin bp 100to 270, a cytomegalovirus early promoter enhancer, the polyoma enhanceron the late side of the replication origin, and adenovirus enhancers.

In other embodiments, the expression vector also contains a ribosomebinding site for translation initiation and a transcription terminator.In still other embodiments of the present invention, the vector may alsoinclude appropriate sequences for amplifying expression.

B. Host Cells for Production of OFQR

In a further embodiment, the present invention provides host cellscontaining the above-described constructs. In some embodiments of thepresent invention, the host cell is a higher eukaryotic cell (e.g., amammalian or insect cell). In other embodiments of the presentinvention, the host cell is a lower eukaryotic cell (e.g., a yeastcell). In still other embodiments of the present invention, the hostcell can be a prokaryotic cell (e.g., a bacterial cell). Specificexamples of host cells include, but are not limited to, Escherichiacoli, Salmonella typhimurium, Bacillus subtilis, and various specieswithin the genera Pseudomonas, Streptomyces, and Staphylococcus, as wellas Saccharomycees cerivisiae, Schizosaccharomycees pombe, Drosophila S2cells, Spodoptera Sf9 cells, Chinese hamster ovary (CHO) cells, COS-7lines of monkey kidney fibroblasts, (Gluzman, Cell 23:175 [1981]), C127, 3T3, 293, 293T, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. In someembodiments, introduction of the construct into the host cell can beaccomplished by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (See e.g., Davis et al., Basic Methodsin Molecular Biology, [1986]). Alternatively, in some embodiments of thepresent invention, the polypeptides of the invention can besynthetically produced by conventional peptide synthesizers.

Proteins can be expressed in mammalian cells, yeast, bacteria, or othercells under the control of appropriate promoters. Cell-free translationsystems can also be employed to produce such proteins using RNAs (e.g.,splice variants of OFQR) derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989).

In some embodiments of the present invention, following transformationof a suitable host strain and growth of the host strain to anappropriate cell density, the selected promoter is induced byappropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period.

C. Fusion Proteins Containing OFQR

The present invention also provides fusion proteins incorporating all orpart of an OFQR polypeptide. Accordingly, in some embodiments of thepresent invention, a coding sequence for one of the polypeptides of thepresent invention can be incorporated as a part of a fusion geneincluding a nucleotide sequence encoding a different polypeptide. It iscontemplated that this type of expression system will find use underconditions where it is desirable to produce an immunogenic fragment ofan OFQR polypeptide. In some embodiments of the present invention, theVP6 capsid protein of rotavirus is used as an immunologic carrierprotein for portions of a OFQR polypeptide of the present invention(e.g., SEQ ID Nos:11, 13, 15, 17 and 23), either in the monomeric formor in the form of a viral particle. In other embodiments of the presentinvention, the nucleic acid sequences corresponding to the portion ofOFQR against which antibodies are to be raised can be incorporated intoa fusion gene construct which includes coding sequences for a latevaccinia virus structural protein to produce a set of recombinantviruses expressing fusion proteins comprising a portion of OFQR as partof the virion. It has been demonstrated with the use of immunogenicfusion proteins utilizing the hepatitis B surface antigen fusionproteins that recombinant hepatitis B virions can be utilized in thisrole as well. Similarly, in other embodiments of the present invention,chimeric constructs coding for fusion proteins containing a portion ofOFQR and the poliovirus capsid protein are created to enhanceimmunogenicity of the set of polypeptide antigens (See e.g., EPPublication No. 025949; and Evans et al., Nature 339:385 [1989]; Huanget al., J. Virol., 62:3855 [1988]; and Schlienger et al., J. Virol.,66:2 [1992]).

In still other embodiments of the present invention, the multipleantigen peptide system for peptide-based immunization can be utilized.In this system, a desired portion of OFQR is obtained directly fromorgano-chemical synthesis of the peptide onto an oligomeric branchinglysine core (see e.g., Posnett et al., J. Biol. Chem., 263:1719 [1988];and Nardelli et al., J. Immunol., 148:914 [1992]). In other embodimentsof the present invention, antigenic determinants of the OFQR proteinscan also be expressed and presented by bacterial cells.

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment ofthe present invention, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, in other embodiments of the present invention, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed to generate a chimeric genesequence (See e.g., Current Protocols in Molecular Biology, supra).

D. Variants of OFQR

Still other embodiments of the present invention provide mutant orvariant forms of OFQR (i.e., muteins). It is possible to modify thestructure of a polypeptide having an activity of OFQR for such purposesas enhancing therapeutic or prophylactic efficacy, or stability (e.g.,ex vivo shelf life, and/or resistance to proteolytic degradation invivo). Such modified polypeptides are considered functional equivalentsof polypeptides having an activity of the subject OFQR proteins asdefined herein. A modified polypeptide can be produced in which theamino acid sequence has been altered, such as by amino acidsubstitution, deletion, or addition.

Moreover, as described above, variant forms (e.g., mutants) of thesubject OFQR proteins are also contemplated as being equivalent to thosepeptides and DNA molecules that are set forth in more detail. Forexample, as described above, the present invention encompasses mutantand variant proteins that contain conservative or non-conservative aminoacid substitutions.

This invention further contemplates a method of generating sets ofcombinatorial mutants of the present OFQR proteins, as well astruncation mutants, and is especially useful for identifying potentialvariant sequences (i.e., homologs) that are functional in binding toOrphanin FQ or other neurotransmitter peptides. The purpose of screeningsuch combinatorial libraries is to generate, for example, novel OFQRhomologs express differential ligand binding properites, oralternatively, possess novel activities all together.

In some embodiments of the combinatorial mutagenesis approach of thepresent invention, the amino acid sequences for a population of OFQRhomologs or other related proteins are aligned, preferably to promotethe highest homology possible. Such a population of variants caninclude, for example, OFQR homologs from one or more species, or OFQRhomologs from the same species but which differ due to mutation orsplice variation. Amino acids that appear at each position of thealigned sequences are selected to create a degenerate set ofcombinatorial sequences.

In a preferred embodiment of the present invention, the combinatorialOFQR library is produced by way of a degenerate library of genesencoding a library of polypeptides which each include at least a portionof potential OFQR protein sequences. For example, a mixture of syntheticoligonucleotides can be enzymatically ligated into gene sequences suchthat the degenerate set of potential OFQR sequences are expressible asindividual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display) containing the set of OFQR sequencestherein.

There are many ways by which the library of potential OFQR homologs canbe generated from a degenerate oligonucleotide sequence. In someembodiments, chemical synthesis of a degenerate gene sequence is carriedout in an automatic DNA synthesizer, and the synthetic genes are ligatedinto an appropriate gene for expression. The purpose of a degenerate setof genes is to provide, in one mixture, all of the sequences encodingthe desired set of potential OFQR sequences. The synthesis of degenerateoligonucleotides is well known in the art (See e.g., Narang, TetrahedronLett., 39:3 9 [1983]; Itakura et al., Recombinant DNA, in Walton (ed.),Proceedings of the 3rd Cleveland Symposium on Macromolecules, Elsevier,Amsterdam, pp 273–289 [1981]; Itakura et al., Annu. Rev. Biochem.,53:323 [1984]; Itakura et al., Science 198:1056 [1984]; Ike et al.,Nucl. Acid Res., 11:477 [1983]). Such techniques have been employed inthe directed evolution of other proteins (See e.g., Scott et al.,Science 249:386–390 [1980]; Roberts et al., Proc. Natl. Acad. Sci. USA89:2429–2433 [1992]; Devlin et al., Science 249: 404–406 [1990]; Cwirlaet al., Proc. Natl. Acad. Sci. USA 87: 6378–6382 [1990]; as well as U.S.Pat. Nos. 5,223,409, 5,198,346, and 5,096,815, each of which isincorporated herein by reference).

It is contemplated that the OFQR nucleic acids (e.g., SEQ ID NOs: 9, 10,12, 14, 16, 18, 19, 20, and 21), and fragments and variants thereof) canbe utilized as starting nucleic acids for directed evolution. Thesetechniques can be utilized to develop OFQR variants having desirableproperties such as increased or decreased binding affinity for OrphaninFQ.

In some embodiments, artificial evolution is performed by randommutagenesis (e.g., by utilizing error-prone PCR to introduce randommutations into a given coding sequence). This method requires that thefrequency of mutation be finely tuned. As a general rule, beneficialmutations are rare, while deleterious mutations are common. This isbecause the combination of a deleterious mutation and a beneficialmutation often results in an inactive protein. The ideal number of basesubstitutions for targeted gene is usually between 1.5 and 5 (Moore andArnold, Nat. Biotech., 14, 458–67 [1996]; Leung et al., Technique,1:11–15 [1989]; Eckert and Kunkel, PCR Methods Appl., 1:17–24 [1991];Caldwell and Joyce, PCR Methods Appl., 2:28–33 (1992); and Zhao andArnold, Nuc. Acids. Res., 25:1307–08 [1997]). After mutagenesis, theresulting clones are selected for desirable activity (e.g., screened forOrphanin FQ binding). Successive rounds of mutagenesis and selection areoften necessary to develop enzymes with desirable properties. It shouldbe noted that only the useful mutations are carried over to the nextround of mutagenesis.

In other embodiments of the present invention, the polynucleotides ofthe present invention are used in gene shuffling or sexual PCRprocedures (e.g., Smith, Nature, 370:324–25 [1994]; U.S. Pat. Nos.5,837,458; 5,830,721; 5,811,238; 5,733,731; all of which are hereinincorporated by reference). Gene shuffling involves random fragmentationof several mutant DNAs followed by their reassembly by PCR into fulllength molecules. Examples of various gene shuffling procedures include,but are not limited to, assembly following DNase treatment, thestaggered extension process (STEP), and random priming in vitrorecombination. In the DNase mediated method, DNA segments isolated froma pool of positive mutants are cleaved into random fragments with DNaseIand subjected to multiple rounds of PCR with no added primer. Thelengths of random fragments approach that of the uncleaved segment asthe PCR cycles proceed, resulting in mutations in present in differentclones becoming mixed and accumulating in some of the resultingsequences. Multiple cycles of selection and shuffling have led to thefunctional enhancement of several enzymes (Stemmer, Nature, 370:398–91[1994]; Stemmer, Proc. Natl. Acad. Sci. USA, 91, 10747–51 [1994];Crameri et al., Nat. Biotech., 14:315–19 [1996]; Zhang et al., Proc.Natl. Acad. Sci. USA, 94:4504–09 [1997]; and Crameri et al., Nat.Biotech., 15:436–38 [1997]). Variants produced by directed evolution canbe screened for ligand binding using any suitable method, including butnot limited to, those disclosed herein.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations, and forscreening cDNA libraries for gene products having a certain property.Such techniques will be generally adaptable for rapid screening of thegene libraries generated by the combinatorial mutagenesis orrecombination of OFQR homologs. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected.

E. Chemical Synthesis of OFQR

In an alternate embodiment of the invention, the coding sequence of anOFQR polypeptide is synthesized, whole or in part, using chemicalmethods well known in the art (See e.g., Caruthers et al., Nucl. AcidsRes. Symp. Ser., 7:215–233 [1980]; Crea and Horn, Nucl. Acids Res.,9:2331 [1980]; Matteucci and Caruthers, Tetrahedron Lett., 21:719[1980]; and Chow and Kempe, Nucl. Acids Res., 9:2807–2817 [1981]). Inother embodiments of the present invention, the protein itself isproduced using chemical methods to synthesize either an entire OFQRamino acid sequence (e.g., SEQ ID Nos: 11, 13, 15, and 17) or a portionthereof. For example, peptides can be synthesized by solid phasetechniques, cleaved from the resin, and purified by preparative highperformance liquid chromatography (See e.g., Creighton, ProteinsStructures And Molecular Principles, W H Freeman and Co, New York N.Y.[1983]). In other embodiments of the present invention, the compositionof the synthetic peptides is confirmed by amino acid analysis orsequencing (See e.g., Creighton, supra).

Direct peptide synthesis can be performed using various solid-phasetechniques (Roberge et al., Science 269:202–204 [1995]) and automatedsynthesis may be achieved, for example, using ABI 431A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer. Additionally, an amino acid sequence of OFQR, orany part thereof, may be altered during direct synthesis and/or combinedusing chemical methods with other sequences to produce a variantpolypeptide.

III. Generation of OFQR Antibodies

In some embodiments, the present invention provides antibodies specificto a OFQR polypeptide. Antibodies can be generated to allow for thedetection of OFQR protein. The antibodies may be prepared using variousimmunogens. In one embodiment, an OFQR splice variant peptide is used asan immunogen in order to generate antibodies that recognize a specificsplice variant of OFQR. Such antibodies include, but are not limited topolyclonal, monoclonal, chimeric, single chain, Fab fragments, and Fabexpression libraries.

Various procedures known in the art may be used for the production ofpolyclonal antibodies directed against an OFQR splice variant. For theproduction of antibody, various host animals can be immunized byinjection with the peptide corresponding to the OFQR epitope includingbut not limited to rabbits, mice, rats, sheep, goats, etc. In apreferred embodiment, the peptide is conjugated to an immunogeniccarrier (e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyholelimpet hemocyanin (KLH)). Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels (e.g.,aluminum hydroxide), surface active substances (e.g., lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (Bacille Calmette-Guerin) and Corynebacterium parvum).

For preparation of monoclonal antibodies directed toward an OFQRpolypeptide, it is contemplated that any technique that provides for theproduction of antibody molecules by continuous cell lines in culturewill find use with the present invention (See e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.). These include, but are not limited to, thehybridoma technique originally developed by Köhler and Milstein (Köhlerand Milstein, Nature 256:495–497 [1975]), as well as the triomatechnique, the human B-cell hybridoma technique (See e.g., Kozbor etal., Immunol. Tod., 4:72 [1983]), and the EBV-hybridoma technique toproduce human monoclonal antibodies (Cole et al., in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77–96 [1985]).

In an additional embodiment of the invention, monoclonal antibodies areproduced in germ-free animals utilizing technology such as thatdescribed in PCT/US90/02545). Furthermore, it is contemplated that humanantibodies will be generated by human hybridomas (Cote et al., Proc.Natl. Acad. Sci. USA 80:2026–2030 [1983]) or by transforming human Bcells with EBV virus in vitro (Cole et al., in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, pp. 77–96 [1985]).

In addition, it is contemplated that techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778; hereinincorporated by reference) will find use in producing OFQR specificsingle chain antibodies. An additional embodiment of the inventionutilizes the techniques described for the construction of Fab expressionlibraries (Huse et al., Science 246:1275–1281 [1989]) to allow rapid andeasy identification of monoclonal Fab fragments with the desiredspecificity for a specific OFQR polypeptide.

It is contemplated that any technique suitable for producing antibodyfragments will find use in generating antibody fragments that containthe idiotype (antigen binding region) of the antibody molecule. Forexample, such fragments include but are not limited to: F(ab′)2 fragmentthat can be produced by pepsin digestion of the antibody molecule; Fab′fragments that can be generated by reducing the disulfide bridges of theF(ab′)2 fragment, and Fab fragments that can be generated by treatingthe antibody molecule with papain and a reducing agent.

In the production of antibodies, it is contemplated that screening forthe desired antibody will be accomplished by techniques known in the art(e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),“sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitation reactions, immunodiffusion assays, in situ immunoassays(e.g., using colloidal gold, enzyme or radioisotope labels, forexample), Western blots, precipitation reactions, agglutination assays(e.g., gel agglutination assays, hemagglutination assays, etc.),complement fixation assays, immunofluorescence assays, protein A assays,and immunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many means are known in the art for detecting binding in animmunoassay and are within the scope of the present invention. As iswell known in the art, the immunogenic peptide should be provided freeof the carrier molecule used in any immunization protocol. For example,if the peptide was conjugated to KLH, it may be conjugated to BSA, orused directly, in a screening assay.

The foregoing antibodies can be used in methods known in the artrelating to the localization and structure of an OFQR polypeptide (e.g.,for Western blotting), measuring levels thereof in appropriatebiological samples, etc. The antibodies can be used to detect OFQR in aspecific tissue (e.g., brain or gastrointestinal tissue) or as OFQRdirected therapeutics (e.g., agonists or antagonists).

The biological tissue samples can then be tested directly for thepresence of an OFQR polypeptide using an appropriate strategy (e.g.,ELISA or radioimmunoassay) and format (e.g., microwells, dipstick (e.g.,as described in International Patent Publication WO 93/03367), etc.Alternatively, proteins in the sample can be size separated (e.g., bypolyacrylamide gel electrophoresis (PAGE), in the presence or not ofsodium dodecyl sulfate (SDS), and the presence of OFQR detected byimmunoblotting (Western blotting). Immunoblotting techniques aregenerally more effective with antibodies generated against a peptidecorresponding to an epitope of a protein, and hence, are particularlysuited to the present invention.

Another method uses antibodies as agents to alter signal transduction.Specific antibodies that bind to the binding domains of OFQR involved insignalling can be used to inhibit the interaction between the variousproteins and their interaction with other ligands. Antibodies that bindto the complex can also be used therapeutically to inhibit interactionsof the protein complex in the signal transduction pathways leading tothe various physiological and cellular effects of Orphanin FQ. Suchantibodies can also be used diagnostically to measure abnormalexpression of OFQR, or the aberrant formation of protein complexes,which may be indicative of a disease state.

IV. Drug Screening Using OFQR

The present invention provides methods and compositions for using OFQRas a target for screening drugs. In some embodiments, drug screeningassays are used to identify compounds that can alter, for example, thebinding of Orphanin FQ to OFQR. In other embodiments, drug screens areused to identify OFQR agonists and antagonists. In preferredembodiments, one or more splice variants of OFQR are utilized toidentify compounds that are active towards some (e.g., one), but notall, receptor variants. The present invention thus provides methods ofidentifying compounds that are active in specific tissues or organs.

Previous studies have suggested that Orphanin FQ modulates a variety ofbiological functions including nociception (pain), food intake, memoryprocesses, caridovacular and renal function, spontaintaneous locomotoractivity, gastrointestinal motility, anxiety and neurotransmitterrelease (Calo et al., Br. J. Pharmacol 129:1261 [2000]). Specifically,Orphanin FQ reduces elementary stress-induced physiological responsessuch as analgesia in rodents and attenuates elaborate behavioral fearresponses elicited when animal are exposed to stressful/anxiogenicsituations (Jenck et al., PNAS 97:4938 [2000]; Koster et al., PNAS96:10444). Orphanin FQ has also been found to play a role in painperception by the measurement of thermal hyperalgesia in rats followingnerve injury (Yamamoto and Nozaki-Taguchi, Anesthesiology 87:1145[1997]). Orphanin FQ has further been shown to play a role ingastrointestinal motility in rats (Yazdani et al., Gastroenterology,116:108 [1999]).

The present invention is not limited to a particular mechanism. Indeed,an understanding of the mechanism is not necessary to practice thepresent invention. Nonetheless, it is contemplated that the diverseeffects of Orphanin FQ in a variety of tissues are due to thepreferential expression of different splice variants in differenttissues (See Example 4). Thus, it is contemplated that OFQR antagonistsor agonists can be targeted to specific tissues and physiologicalresponses (e.g., pain, gastrointestinal motility, learning and memory,drug dependence, and stress-related neuronal dysfunctions).

In addition, further experiments conducted during the course ofdevelopment of the present invention provided a role for Orphanin FQpeptide in the regulation of colonic motility (See e.g., Experimentalsection below). Accordingly, in some embodiments, Orphanin FQ peptidesor mimetics or variants thereof are utilized as candidates in drugscreening assays. In other embodiments, such compositions find use inresearch applications such as including, but not limited to, the studyof colonic motility and the identification of compounds useful intreating disorders of colonic motility.

The present invention is not limited to any one drug-screening method.Indeed, any suitable method may be utilized. In some embodiments, drugscreening is carried out using the method described in U.S. Pat. No.6,172,075 (herein incorporated by reference). Briefly, the cDNA for aparticular splice variant of OFQR is cloned into an expression vector(e.g., including, but not limited to, those described above) andexpressed in a mammalian cell line (e.g., including, but not limited to,those described above). In some embodiments, greater than one (e.g.,two) OFQR receptors are expressed in the same cell (See e.g., U.S. Pat.No. 5,976,807; herein incorporate by reference).

In some embodiments, binding assays are performed with intact cellsexpressing OFQR receptor on their surfaces (the presence of OFQR on cellsurfaces can be determined, for example, by one of the immunologicalmethods described above). In other embodiments, membrane fractions areisolated from the cells (See e.g., U.S. Pat. No. 6,172,075).

Binding assays are next performed using any suitable method. Forexample, in some embodiments, filter binding assays utilizingradiolabelled OFQR are used to assay the binding of a ligand to OFQR(See e.g., U.S. Pat. No. 6,172,075; herein incorporated by reference).In other embodiments, binding is assayed by ligand binding associatedinhibition of forskolin-stimulated camp accumulation (See e.g., U.S.Pat. No. 5,821,219; herein incorporated by reference). In still otherembodiments, a reporter gene expression assay is utilized in which cellsare transformed with a plasmid containing an OFQR gene and a secondplasmid containing a reporter plasmid (e.g., cAMP responsive element)(See e.g., Wnendt et al., Mol. Pharmacol. 56:334 [1999] and U.S. Pat.No. 5,976,807; herein incorporated by reference for exemplary methods).

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to OFQR and is describedin detail in WO 84/03564, incorporated herein by reference. Briefly,large numbers of different small peptide test compounds are synthesizedon a solid substrate, such as plastic pins or some other surface. Thepeptide test compounds are then reacted with OFQR and washed. Bound OFQRpolypeptides are then detected by methods well known in the art.

Another technique uses OFQR antibodies, generated as discussed above.Such antibodies capable of binding to specific OFQR splice variantscompete with a test compound for binding to an OFQR polypeptide. In thismanner, the antibodies can be used to detect the presence of any peptidethat shares one or more antigenic determinants of a specific OFQRpolypeptide.

The present invention contemplates many other means of screeningcompounds. The examples provided above are presented merely toillustrate a range of techniques available. One of ordinary skill in theart will appreciate that many other screening methods can be used.

In particular, the present invention contemplates the use of cell linestransfected with OFQR splice variants and variants or mutants thereoffor screening compounds for activity, and in particular to highthroughput screening of compounds from combinatorial libraries (e.g.,libraries containing greater than 10⁴ compounds). The cell lines of thepresent invention can be used in a variety of screening methods. In someembodiments, the cells can be used in second messenger assays thatmonitor signal transduction following activation of cell-surfacereceptors. In other embodiments, the cells can be used in reporter geneassays that monitor cellular responses at the transcription/translationlevel. In still further embodiments, the cells can be used in cellproliferation assays to monitor the overall growth/no growth response ofcells to external stimuli.

In second messenger assays, the host cells are preferably transfected asdescribed above with vectors encoding OFQR splice variants or variantsor mutants thereof. The host cells are then treated with a compound orplurality of compounds (e.g., from a combinatorial library) and assayedfor the presence or absence of a response. It is contemplated that atleast some of the compounds in the combinatorial library can serve asagonists, antagonists, activators, or inhibitors of the protein orproteins encoded by the vectors. It is also contemplated that at leastsome of the compounds in the combinatorial library can serve asagonists, antagonists, activators, or inhibitors of protein actingupstream or downstream of the protein encoded by the vector in a signaltransduction pathway.

In some embodiments, the second messenger assays measure fluorescentsignals from reporter molecules that respond to intracellular changes(e.g., Ca²⁺ concentration, membrane potential, pH, IP₃, cAMP,arachidonic acid release) due to stimulation of membrane receptors andion channels (e.g., ligand gated ion channels; see Denyer et al., DrugDiscov. Today 3:323–32 [1998]; and Gonzales et al., Drug. Discov. Today4:431–39 [1999]). Examples of reporter molecules include, but are notlimited to, FRET (florescence resonance energy transfer) systems (e.g.,Cuo-lipids and oxonols, EDAN/DABCYL), calcium sensitive indicators(e.g., Fluo-3, FURA 2, INDO 1, and FLUO3/AM, BAPTA AM),chloride-sensitive indicators (e.g., SPQ, SPA), potassium-sensitiveindicators (e.g., PBFI), sodium-sensitive indicators (e.g., SBFI), andpH sensitive indicators (e.g., BCECF).

In general, the host cells are loaded with the indicator prior toexposure to the compound. Responses of the host cells to treatment withthe compounds can be detected by methods known in the art, including,but not limited to, fluorescence microscopy, confocal microscopy (e.g.,FCS systems), flow cytometry, microfluidic devices, FLIPR systems (See,e.g., Schroeder and Neagle, J. Biomol. Screening 1:75–80 [1996]), andplate-reading systems. In some preferred embodiments, the response(e.g., increase in fluorescent intensity) caused by compound of unknownactivity is compared to the response generated by a known agonist andexpressed as a percentage of the maximal response of the known agonist.The maximum response caused by a known agonist is defined as a 100%response. Likewise, the maximal response recorded after addition of anagonist to a sample containing a known or test antagonist is detectablylower than the 100% response.

The cells are also useful in reporter gene assays (e.g., as describedabove). Reporter gene assays involve the use of host cells transfectedwith vectors encoding a nucleic acid comprising transcriptional controlelements of a target gene (i.e., a gene that controls the biologicalexpression and function of a disease target) spliced to a codingsequence for a reporter gene. Therefore, activation of the target generesults in activation of the reporter gene product.

Examples of reporter genes finding use in the present invention include,but are not limited to, chloramphenicol transferase, alkalinephosphatase, firefly and bacterial luciferases, β-galactosidase,β-lactamase, and green fluorescent protein. The production of theseproteins, with the exception of green fluorescent protein, is detectedthrough the use of chemiluminescent, calorimetric, or bioluminecentproducts of specific substrates (e.g., X-gal and luciferin). Comparisonsbetween compounds of known and unknown activities may be conducted asdescribed above.

In some embodiments, drug screening assays are performed (e.g., usingone of the methods described above) in which multiple splice variants(e.g., two or more) of OFQR are simultaneously assayed. The variants canbe expressed in one cell line, or alternatively, in a series of membranepreparations. In such an analysis, the comparative effect of multiplecompounds can be assayed. The ability of compounds to compete with eachother and/or the endogenous ligand (e.g., Orphanin FQ) provides datauseful in identifying potential therapeutics.

V. Therapeutic Compositions

In some embodiments, the present invention provides therapeuticcompositions directed towards the Orphanin FQ receptor. In someembodiments, the compositons are peptides, modified peptides, or peptidemimetics. In some embodiments, the peptides or peptide mimetics areOrphanin FQ peptides or derivatives or mimetics thereof. In otherembodiments, the therapeutics are small molecules.

As described below (See e.g., experimental section), the administrationof Orphanin FQ to an animal model of postoperative ileus resulted in anincrease in colonic transit time. Accordingly, in some embodiments, thestructure of the Orphanin FQ peptide is used as a starting point in thedesign of peptide and small molecule mimetics for use in the treatmentof disorders of colonic transit.

In certain embodiments, the therapeutics of the present invention finduse in the treatment of disorders of colonic transit (e.g., constipation(e.g., encopresis), colonic inertia, diarrhia disorders, irritable boweldisorder (IBD), mega colon, colonic pseudo obstruction, and postoperative ileus).

A. Protease Resistant Peptides

In some embodiments, the peptide-based therapeutics of the presentinvention are protease resistant. In one embodiment, suchprotease-resistant peptides are peptides comprising protecting groups.In a preferred embodiment, the present invention contemplates a peptideof the present invention or variant thereof that is protected fromexoproteinase degradation by N-terminal acetylation (“Ac”) andC-terminal amidation. Such peptides are useful for in vivoadministration because of their resistance to proteolysis.

In another embodiment, the present invention also contemplates peptidesprotected from endoprotease degradation by the substitution of L-aminoacids in the peptides with their corresponding D-isomers. It is notintended that the present invention be limited to particular amino acidsand particular D-isomers. This embodiment is feasible for all aminoacids, except glycine; that is to say, it is feasible for all aminoacids that have two stereoisomeric forms. By convention thesemirror-image structures are called the D and L forms of the amino acid.These forms cannot be interconverted without breaking a chemical bond.With rare exceptions, only the L forms of amino acids are found innaturally occurring proteins.

B. Mimetics

In still further embodiments, compounds mimicking the necessaryconformation for recognition and docking to the Orphanin FQ receptor arecontemplated as therapeutics of the present invention. A variety ofdesigns for such mimetics are possible. For example, cyclic-containingpeptides, in which the necessary conformation for binding is stabilizedby nonpeptides, are specifically contemplated. U.S. Pat. Nos. 5,192,746,5,169,862, 5,539,085, 5,576,423, 5,051,448, and 5,559,103, all herebyincorporated by reference, describe multiple methods for creating suchcompounds.

Synthesis of nonpeptide compounds that mimic peptide sequences is alsoknown in the art. Eldred et al. (J. Med. Chem., 37:3882 [1994]) describenonpeptide antagonists that mimic the Arg-Gly-Asp sequence. Likewise, Kuet al. (J. Med. Chem., 38:9 [1995]) give further elucidation of thesynthesis of a series of such compounds. Such nonpeptide compounds thatmimic peptide inhibitors of the present invention are specificallycontemplated by the present invention.

The present invention also contemplates synthetic mimicking compoundsthat are multimeric compounds that repeat the relevant peptide sequence.As is known in the art, peptides can be synthesized by linking an aminogroup to a carboxyl group that has been activated by reaction with acoupling agent, such as dicyclohexylcarbodiimide (DCC). The attack of afree amino group on the activated carboxyl leads to the formation of apeptide bond and the release of dicyclohexylurea. It can be necessary toprotect potentially reactive groups other than the amino and carboxylgroups intended to react.

For example, the α-amino group of the component containing the activatedcarboxyl group can be blocked with a tertbutyloxycarbonyl group. Thisprotecting group can be subsequently removed by exposing the peptide todilute acid, which leaves peptide bonds intact.

With this method, peptides can be readily synthesized by a solid phasemethod by adding amino acids stepwise to a growing peptide chain that islinked to an insoluble matrix, such as polystyrene beads. Thecarboxyl-terminal amino acid (with an amino protecting group) of thedesired peptide sequence is first anchored to the polystyrene beads. Theprotecting group of the amino acid is then removed. The next amino acid(with the protecting group) is added with the coupling agent. This isfollowed by a washing cycle. The cycle is repeated as necessary.

In one embodiment, the mimetics of the present invention are peptideshaving sequence homology to the peptides described herein (including,but not limited to, peptides in which L-amino acids are replaced bytheir D-isomers). One common methodology for evaluating sequencehomology, and more importantly statistically significant similarities,is to use a Monte Carlo analysis using an algorithm written by Lipmanand Pearson to obtain a Z value. According to this analysis, a Z valuegreater than 6 indicates probable significance, and a Z value greaterthan 10 is considered to be statistically significant (Pearson andLipman, Proc. Natl. Acad. Sci. (USA), 85:2444–2448 (1988); Lipman andPearson, Science, 227:1435 (1985)). In the present invention, syntheticpolypeptides useful in inhibiting RGS proteins are those peptides withstatistically significant sequence homology and similarity (Z value ofLipman and Pearson algorithm in Monte Carlo analysis exceeding 6).

In some particularly preferred embodiments, mimetic compounds are thosein which the peptide cycle is replaced by any non-peptide scaffold thatallows comparable positioning of functional equivalents of the K, T, andE sidechains of the peptide inhibitors.

C. Other Modified Peptides

The present invention further includes peptides modified to improve oneor more properties useful in pharmaceutical compounds. For example, insome embodiments, peptides are modified to enhance their ability toenter intracellular space. Such modifications include, but are notlimited to, the addition of charged groups, lipids and myristate groups(See e.g., U.S. Pat. No. 5,607,691; herein incorporated by reference).

In other embodiments, the peptides of the present invention may be inthe form of a liposome in which isolated peptide is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers which inaqueous solution. Suitable lipids for liposomal formulation include,without limitation, monoglycerides, diglycerides, sulfatides,lysolecithin, phospholipids, saponin, bile acids, and the like.Preparation of such liposomal formulations is within the level of skillin the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871;4,501,728; 4,837,028; and 4,737,323, all of which are incorporatedherein by reference.

VI. Pharmaceutical Compositions

The present invention further provides pharmaceutical compositions(e.g., those developed by the drug screening applications describedabove) which may comprise agonists or antagonists of OFQR, includingantibodies, peptides, antisense oligonucleotides, and small moleculecompounds, alone or in combination with at least one other agent, suchas a stabilizing compound, and may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water.

The methods of the present invention find use in treating diseases oraltering physiological states characterized by Orphanin FQ/OFQRsignaling. Consequently, compounds developed using the methods of thepresent invention are useful in the treatment of psychiatric,neurological and physiological disorders, especially, but not limitedto, amelioration of symptoms of anxiety and stress disorders,depression, trauma, memory loss due to Alzheimer's disease or otherdementias, epilepsy and convulsions, acute and/or chronic painconditions, symptoms of addictive drug withdrawal, control of waterbalance, Na⁺ excretion, arterial blood pressure disorders, colonictransit disorders and metabolic disorders such as obesity.

The formulations developed by the methods of the present invention areuseful for parenteral administration, such as intravenous, subcutaneous,intramuscular, and intraperitoneal. As is well known in the medicalarts, dosages for any one patient depends upon many factors, includingthe patient's size, body surface area, age, the particular compound tobe administered, sex, time and route of administration, general health,and interaction with other drugs being concurrently administered.

Accordingly, in some embodiments of the present invention, compoundsdeveloped by the drug screening methods of the present invention can beadministered to a patient alone, or in combination with nucleotidesequences, drugs or hormones or in pharmaceutical compositions where itis mixed with excipient(s) or other pharmaceutically acceptablecarriers. In one embodiment of the present invention, thepharmaceutically acceptable carrier is pharmaceutically inert. Inanother embodiment of the present invention, compounds may beadministered alone to individuals subject to or suffering from adisease.

Depending on the condition being treated, these pharmaceuticalcompositions may be formulated and administered systemically or locally.Techniques for formulation and administration may be found in the latestedition of “Remington's Pharmaceutical Sciences” (Mack Publishing Co,Easton Pa.). Suitable routes may, for example, include oral ortransmucosal administration; as well as parenteral delivery, includingintramuscular, subcutaneous, intramedullary, intrathecal,intraventricular, intravenous, intraperitoneal, or intranasaladministration.

For injection, the pharmaceutical compositions of the invention may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. For tissue or cellular administration,penetrants appropriate to the particular barrier to be permeated areused in the formulation. Such penetrants are generally known in the art.

In other embodiments, the pharmaceutical compositions of the presentinvention can be formulated using pharmaceutically acceptable carrierswell known in the art in dosages suitable for oral administration. Suchcarriers enable the pharmaceutical compositions to be formulated astablets, pills, capsules, liquids, gels, syrups, slurries, suspensionsand the like, for oral or nasal ingestion by a patient to be treated.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. For example, aneffective amount of composition may be that amount that results insubstantial elimination of symptoms characteristic of the disease statebeing treated. Determination of effective amounts is well within thecapability of those skilled in the art, especially in light of thedisclosure provided herein.

In addition to the active ingredients these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known (e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes).

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are carbohydrate or protein fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; starch from corn,wheat, rice, potato, etc; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; andgums including arabic and tragacanth; and proteins such as gelatin andcollagen. If desired, disintegrating or solubilizing agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, (i.e., dosage).

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients mixed with fillers orbinders such as lactose or starches, lubricants such as talc ormagnesium stearate, and, optionally, stabilizers. In soft capsules, theactive compounds may be dissolved or suspended in suitable liquids, suchas fatty oils, liquid paraffin, or liquid polyethylene glycol with orwithout stabilizers.

Compositions comprising a compound of the invention formulated in apharmaceutical acceptable carrier may be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM–50 mM histidine, 0.1%–2% sucrose,2%–7% mannitol at a pH range of 4.5 to 5.5 that is combined with bufferprior to use.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. Then, preferably, dosage can be formulated in animalmodels (particularly murine models) to achieve a desirable circulatingconcentration range that adjusts the level of the compound.

A therapeutically effective dose refers to that amount of the compounddeveloped by the methods of the present invention that amelioratessymptoms of the disease state. Toxicity and therapeutic efficacy of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index, and itcan be expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit largetherapeutic indices are preferred. The data obtained from these cellculture assays and additional animal studies can be used in formulatinga range of dosage for human use. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage varies within this rangedepending upon the dosage form employed, sensitivity of the patient, andthe route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors which may be taken into account include theseverity of the disease state; age, weight, and gender of the patient;diet, time and frequency of administration, drug combination(s),reaction sensitivities, and tolerance/response to therapy. Long actingpharmaceutical compositions might be administered every 3 to 4 days,every week, or once every two weeks depending on half-life and clearancerate of the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature (See, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212,all of which are herein incorporated by reference). Administration tothe bone marrow may necessitate delivery in a manner different fromintravenous injections.

VI. Transgenic Animals Expressing Exogenous OFQR Genes and SpliceVariants, Homologs, Mutants, and Variants Thereof

The present invention contemplates the generation of transgenic animalscomprising an exogenous OFQR gene or splice variants, homologs, mutants,or variants thereof. In preferred embodiments, the transgenic animaldisplays an altered phenotype as compared to wild-type animals. In someembodiments, the altered phenotype is the overexpression of mRNA for anOFQR gene (e.g., a splice variant) as compared to wild-type levels ofOFQR expression. In other embodiments, the altered phenotype is thedecreased expression of mRNA for an endogenous OFQR gene (e.g., a splicevariant) gene as compared to wild-type levels of endogenous OFQRexpression. Methods for analyzing the presence or absence of suchphenotypes include Northern blotting, mRNA protection assays, andRT-PCR. In other embodiments, the transgenic animals have a knock outmutation of one or more variants of the OFQR gene (e.g., including butnot limited to, splice variants). In still further embodiments,transgenic animals express an OFQR mutant gene. Since the specifictissue where different variants of OFQR are known, mutations, knockouts,etc. can be targeted to a specific tissue in order to study thelocalized effect of Orphanin FQ/OFQR signaling.

In some embodiments, the transgenic animals of the present inventionfind use in drug screens. In some embodiments, test compounds (e.g., adrug developed by the drug screening methods described above) andcontrol compounds (e.g., a placebo) are administered to the transgenicanimals and the control animals and the effects evaluated.

The transgenic animals can be generated via a variety of methods. Insome embodiments, embryonal cells at various developmental stages areused to introduce transgenes for the production of transgenic animals.Different methods are used depending on the stage of development of theembryonal cell. The zygote is the best target for micro-injection. Inthe mouse, the male pronucleus reaches the size of approximately 20micrometers in diameter, which allows reproducible injection of 1–2picoliters (pl) of DNA solution. The use of zygotes as a target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host genome before the first cleavage(Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438–4442 [1985]). As aconsequence, all cells of the transgenic non-human animal will carry theincorporated transgene. This will in general also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene. U.S. Pat. No.4,873,191 describes a method for the micro-injection of zygotes; thedisclosure of this patent is incorporated herein in its entirety.

In other embodiments, retroviral infection is used to introducetransgenes into a non-human animal. In some embodiments, the retroviralvector is utilized to transfect oocytes by injecting the retroviralvector into the perivitelline space of the oocyte (U.S. Pat. No.6,080,912, incorporated herein by reference). In other embodiments, thedeveloping non-human embryo can be cultured in vitro to the blastocyststage. During this time, the blastomeres can be targets for retroviralinfection (Janenich, Proc. Natl. Acad. Sci. USA 73:1260–1264 [1976]).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Hogan et al., in Manipulatingthe Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. [1986]). The viral vector system used to introduce thetransgene is typically a replication-defective retrovirus carrying thetransgene (D. Jahner et al., Proc. Natl. Acad. Sci. USA 82:6927–693[1985]). Transfection is easily and efficiently obtained by culturingthe blastomeres on a monolayer of virus-producing cells (Van der Putten,supra; Stewart, et al., EMBO J., 6:383–388 [1987]). Alternatively,infection can be performed at a later stage. Virus or virus-producingcells can be injected into the blastocoele (D. Jahner et al., Nature298:623–628 [1982]). Most of the founders will be mosaic for thetransgene since incorporation occurs only in a subset of cells that formthe transgenic animal. Further, the founder may contain variousretroviral insertions of the transgene at different positions in thegenome that generally will segregate in the offspring. In addition, itis also possible to introduce transgenes into the germline, albeit withlow efficiency, by intrauterine retroviral infection of the midgestationembryo (Jahner et al., supra [1982]). Additional means of usingretroviruses or retroviral vectors to create transgenic animals known tothe art involves the micro-injection of retroviral particles ormitomycin C-treated cells producing retrovirus into the perivitellinespace of fertilized eggs or early embryos (PCT International ApplicationWO 90/08832 [1990], and Haskell and Bowen, Mol. Reprod. Dev., 40:386[1995]).

In other embodiments, the transgene is introduced into embryonic stemcells and the transfected stem cells are utilized to form an embryo. EScells are obtained by culturing pre-implantation embryos in vitro underappropriate conditions (Evans et al., Nature 292:154–156 [1981]; Bradleyet al., Nature 309:255–258 [1984]; Gossler et al., Proc. Acad. Sci. USA83:9065–9069 [1986]; and Robertson et al., Nature 322:445–448 [1986]).Transgenes can be efficiently introduced into the ES cells by DNAtransfection by a variety of methods known to the art including calciumphosphate co-precipitation, protoplast or spheroplast fusion,lipofection and DEAE-dextran-mediated transfection. Transgenes may alsobe introduced into ES cells by retrovirus-mediated transduction or bymicro-injection. Such transfected ES cells can thereafter colonize anembryo following their introduction into the blastocoel of ablastocyst-stage embryo and contribute to the germ line of the resultingchimeric animal (for review, See, Jaenisch, Science 240:1468–1474[1988]). Prior to the introduction of transfected ES cells into theblastocoel, the transfected ES cells may be subjected to variousselection protocols to enrich for ES cells which have integrated thetransgene assuming that the transgene provides a means for suchselection. Alternatively, the polymerase chain reaction may be used toscreen for ES cells which have integrated the transgene. This techniqueobviates the need for growth of the transfected ES cells underappropriate selective conditions prior to transfer into the blastocoel.

In still other embodiments, homologous recombination is utilizedknockout gene function or create deletion mutants (e.g., mutants inwhich the OFQR is altered to effect its ligand binding or signalingabilities). Methods for homologous recombination are described in U.S.Pat. No. 5,614,396, incorporated herein by reference.

EXAMPLES

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); μ (micron); M (Molar); μM(micromolar); mM (millimolar); N (Normal); mol (moles); mmol(millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg(milligrams); μg (micrograms); ng (nanograms); L (liters); ml(milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nM (nanomolar); ° C. (degrees Centigrade); and PBS(phosphate buffered saline).

Example 1 Isolation of OFQR Genomic Clones

This example describes the isolation of genomic clones from ratcontaining the OFQR. A rat genomic library in the bacteriophage EMBL-3(Clontech) was screened as previously reported (Blandizzi et al.,Biochem. Biophys. Res. Commun. 202:947 [1994]), using a ³²P-labeled cDNAfragment generated by the polymerase chain reaction (PCR) with primersbased on the cDNA sequences of the rat OFQR (U01913, Bunzow et al., FEBSLett. 347:284 [1994]; and L29419, Wick et al., Brain Res. Mol. BrainRes. 27:37 [1994]). Positive clones were digested with Pst I, Acc I, andSac I. The resulting restriction fragments were subcloned into phageM13mp18 or -mp19 and sequenced in both directions by the dideoxy chaintermination method (Sanger et al., 1977) using the T7 Sequenase Kit(Amersham). Oligonucleotide primers for sequencing or for PCR weresynthesized on a DNA synthesizer (Applied Biosystems-380B). Computeranalysis of nucleotide sequences was performed with the GeneticsComputer Group (GCG) program (Biotechnology Center, University ofWisconsin, Madison, Wis.).

A random-primed ³²P-labeled rat OFQR cDNA probe gave positivehybridization signals with five clones of 10⁶ plaques from a rat genomiclibrary. Two of the genomic clones were overlapped to cover thefull-length sequence of the gene encoding OFQR. The rat OFQR geneexceeded 10 kb in length and contained six exons, which were interruptedby five introns (FIG. 1). All exon/intron boundaries conformed to knownsplice junction consensus sequences (Mount, Nucleic Acids Res. 10:459[1982]) (Table 2). The ATG translation initiation codon was located inexon 2, and the open reading frame consisted of 1283 bp and six exonsranging from 34 to 524 bp (FIG. 2). An mRNA variant containing exon 4(see FIG. 4C for splice variants) is formed by joining nt 2344 (splicedonor G/gt) to nt 2628 (acceptor ag/GC), which results in a threonine(T) residue at position 136 and early termination at nt 2639–2641 (TGA).Splice variants deleted for exons 3 and 4 cause a translationframeshift, avoiding early termination and resulting in the replacementof serine (S) with arginine (R) at position 75 in exon 2, and threonine(T) with histidine (H) at position 136 in exon 5. In these forms, thestop codon (TGA) is located at nt 3586–3588 in exon 6.

Exon 2 encodes the putative extracellular amino terminus andtransmembrane (TM)-spanning region I of the receptor. Exon 5 encodes TMregions II to IV, and exon 6 encodes the remaining TM regions V to VIIand the intracellular carboxyl terminus. The 5′ flanking region of therat OFQR gene (FIG. 2) contains a tandem dinucleotide sequence (GA)₂₆,two (AAAAC)₃ repeat sequences, two potential recognition sites for RNApolymerase II (TATA), and common motifs for transcription factors, suchas Ap2 and Sp1. The OFQR nucleotide sequence, including that of the 5′flanking region (FIG. 2) has been deposited in the GenBank databaseunder the accession number AF216218.

Example 2 Primer Extension Analysis

This Example describes the use of primer extension analysis to determinethe putative transcription initiation sites of the OFQR gene. Primerextension was performed with poly (A)⁺ RNA (500 ng) from rat brain andcolon combined with two end-labeled antisense oligonucletide primers:P18 (5′GCAGCAGCCGGTGCGGCAGCCAG3′; (SEQ ID NO:1) and P42(5′TAGACACCGTCCACTGAGGCC3′; SEQ ID NO:2), as previously described(Muraoka et al., Am. J. Physiol. 271:G1101 [1996]). FIG. 2 shows thelocation of the primers (exon 1 and intron 1, respectively).

When the end-labeled primer P18 was used in the extension reaction, anelongation product of 72 bp was detected in both brain and colon, andthe transcription initiation site was identified as the cytosine 103 bpdownstream from the TATA box at nt 750 in the 5′ flanking region (FIG.3A). In contrast, when primer P42 was used, an elongation product of 71bp was detected in brain tissue only, and the transcription initiationsite was identified as the guanine 133 bp downstream from the TATA boxat nt 139 in intron 1 (FIG. 3B).

Example 3 PCR Cloning of Gene Splice Variants

PCR cloning was performed with poly (A)⁺ RNA (150 ng) from rat brain,colon, and liver using strategically designed oligonucleotide primers(Table 1), as previously reported (Song et al., Proc. Natl. Acad. Sci.USA. 90, 9085 [1993]). Primer P13 (SEQ ID NO:7) in the 3′ untranslatedregion was used in combination with either primer P9 (SEQ ID NO:6) inexon 1 or primer P3 (SEQ ID NO:3) in intron 1 to produce full-lengthcDNAs. Primer P3 containing the 20-mer of intron 1 sequence was designedto verify splice variants that did not express exon 1. The resulting PCRproducts served as the templates for subsequent PCRs using internalprimers P5 (SEQ ID NO:4) and P10 (SEQ ID NO:5). Gene splice variantswere identified by sequencing as described above.

When primers P9 and P13 followed by primers P5 and P10 were used forPCR, multiple bands were obtained from the brain and colon RNA (FIG.4A), rather than the expected single band. Using primers P3 and P13followed by primers P5 and P10, specific bands were demonstrated only inbrain, as shown in FIG. 4B. The sequencing results of these PCR productsrevealed that rat OFQR expressed at least nine splice variants deletedfor exon 1, or exons 3 to 5, as shown in FIG. 5C. Previously reportedcDNAs LC132 (Bunzow et al., FEBS Lett. 347:284 [1994]) and Hyp 8-1 (Wicket al., Brain Res. Mol. Brain Res. 27:37 [1994]) deleted for exon 1 hadthe same sequence as the OFQR-c and OFQR-c′, respectively. Pan et al.,Gene 171:255 [1998] reported that the coding regions of OFQR-b (-b′), -c(-c′), and -d (-d′) splice variants corresponded to the coding regionsof KOR-3a, -3, and -3d, respectively, in mouse brain. Unlike the OFQR-c(-c′) and -d (-d′) isoforms, translation of the OFQR-a, -b (-b′), and -e(-e′) is predicted to result in early termination at exon 5 or 6 due toshifts of the open reading frame (FIG. 4C). We did not detect a splicevariant containing intron 5, as reported by Wang et al., supra. Inaddition, the amino acid sequence deduced by this study varies from thatpublished by Wang et al., supra, as shown in FIG. 4D.

Example 4 Expression of Alternative Splicing in Various Rat Tissues

RT-PCR with poly (A)⁺ RNA (150 ng) from various rat tissues (brain,liver, kidney, heart, lung, spleen, skeletal muscle, testes, esophagus,gastric fundus, corpus, antrum, pylorus, duodenum, jejunum, ileum,proximal colon, and distal colon) was performed using primers P5 and P29(Table 1). The splice variants were analyzed by Southern blot or theScanJet 4c/T (Hewlett Packard). A housekeeping gene, GAPDH, served as aninternal control.

As shown in FIG. 5A, PCR products of OFQR variants were not detected inliver, kidney, heart, lung, spleen, or skeletal muscle, whereas twoforms, OFQR-a and -c, were expressed in testes. These forms, in additionto OFQR-b, were expressed in pylorus, ileum, and proximal colon. Bothforms, OFQR-b and -c (or -d) were expressed in brain and antrum. OFQR-c(or -d) was expressed in esophagus, fundus, corpus, duodenum, jejunum,and descending colon. OFQR-c and -d could not be distinguished in theelectrophoretic analysis of RT-PCR products due to the small sizedifference (i.e., 15 bp). As shown in FIG. 5B, the OFQR-c′ or -d′ splicevariants were expressed only in brain. The sequencing data agree in partwith the results of Wang et al., supra and Osinski et al., Eur. J.Pharmacol. 365, 281 [1999], who used RT-PCR to study the expression ofOFQR in rat and pig tissues, respectively.

TABLE 1 Sequence of Oligonucleotide Primers Used in PCR Name LocationOrientation Sequence (5′–3′) P3  Intron 1/Exon 2 SensegtttctgtgccctgttccagGAACTG (SEQ ID NO:3) P5  Exon 2 SenseCCTGCCCCTTGGACTCAAGGTCACC (SEQ ID NO:4) P9  Exon 1 SenseGCTCAGTCCACTGTGCTCCTGCCTG (SEQ ID NO:6) P10 Exon 6 AntisenseGGTCCACGCCTAGTCATGCTGGCC (SEQ ID NO:5) P13 3′ UTR AntisenseGGTGCTAAAAGGTCTTCCTCTAGGAC (SEQ ID NO:7) P29 Exon 5 AntisenseCAGTGTTAGCAAGACCAGGG (SEQ ID NO:8) Primers P3, P5, and P9 possess aBamHI site at their 5′ end. Primers P10 and P13 possess an EcoRI site attheir5′end. The intron sequence is in lowercase.

TABLE 2 Nucleotide Sequence of Exon-Intron Boundaries in Rat OFQReceptor Gene Sequence of Exon-Intron Junctions Exon Exon Size (bp) (5′Splice Donor . . . Intron (bp) . . . 3′ Splice Acceptor) 1 250 GTTGGAGgtaagagggg . . . (467) . . . ccctgttccag GAACTGT (SEQ ID NO:25) 2 257TCCTCAG gtaggctggg . . . (555) . . . tttttttccag CTGGGAG (SEQ ID NO:26)3 84 ACAGCAG gtgaggactt . . . (1353) . . . ttcattgctag ACAATAC (SEQ IDNO:27) 4 145 ACATTCA gttagatatg . . . (283) . . . gtcctctacag GCACACC(SEQ ID NO:28) 5 358 GATGAAG gtcagtgggt . . . (81) . . . ctctcctgcagAGATCGA (SEQ ID NO:29) 6 524 AGCATGA (SEQ ID NO:30) Exon sequences arein uppercase and intron sequences are in lowercase.

Example 5 Molecular Interactions Between Orphanin FQ and its Receptor

Orphanin FQ is a neuropeptide that structurally resembles an opioidpeptide. The Orphanin FQ receptor (OFQR) exhibits 50–60% sequenceidentity with cloned opioid receptors but does not bind with any knownopioids. While not being limited to any particular mechanism, thepresent invention contemplates that Orphanin FQ stimulates peristalsisand enhances colonic transit by inhibiting purinergic pathways inmyenteric plexus. To delineate the molecular interactions between OFQRand its ligands, Cos-7 cells were transfected with normal or chimericOFQR genes. Binding studies were performed with Orphanin FQ17. Theability of Orphanin FQ and related peptides to inhibit cAMP productionin Cos-7 cells was evaluated. Purified OFQ17 and synthetic OFQ17 withamino acids R8K, K13C, or C-terminal OH converted to NH₂ producedsimilar concentration dependent inhibition of [¹²⁵I] Y14-OFQ17 binding(IC₅₀=10⁻¹⁰ M). OFQ11 was much less efficient (IC₅₀=5×10⁻⁶M) suggestingthat OFQ12 contains amino acid(s) essential for binding. Dynorphin 17and β endorphin 31 had no effect on OFQ binding. Dynorphin encodingnucleic acids and gene products are known in the art, see for example,NM_(—)024411, NM_(—)133828, NM_(—)174139, NM_(—)018863, M10088,AH002816, K02268, K02267, Alo34562, U58500, and U38912. OFQ17dose-dependently inhibits 10⁻⁴M forskolin-stimulated cAMP production(IC₅₀=10⁻¹⁰M) in Cos-7 cells transfected with OFQ receptor gene.Pertussis toxin reversed the inhibitory action of OFQ indicating OFQ-Ris linked to Gi/Go. Specific OFQ binding was observed in Cos-7 cellstransfected with normal OFQ-R gene (NT) (28±14 in nontransfected cellsvs 141±42 in NT, fmol/mg protein). Binding was abolished by pointmutations at D206V and A213V all in the 2^(nd) extracellular loop (2EL),but not by Y207V (2EL), F212V (2EL), RRLR237-240V4 (3^(rd) intracellularloop, 3IL, N-terminal), RRITR 256-269 V5 (3IL, C-terminal), andET292-293 V2 (3^(rd) extracellular loop (3EL) a predicted area foropioid and naloxone binding). NT, Y207V, RRLR237-240V4 (3IL) andET292-293V2 (3EL) did not alter OFQ's inhibitory action on forskolinstimulated cAMP production. In contrast, F212V and RRITR 256-260V5abolished the ability of OFQ to inhibit cAMP without affecting itsbinding. 3D modeling predicts D206 (2EL), which may bind to R8 of OFQ byionic bonds, and A213 (2EL) of the OFQ-R are key amino acids forbinding. On the other hand, F212 which may form Π—Π interactions with F4of OFQ, and RRITR 256-260 (conserved G protein-coupling BBXXB motif inthe Cc-terminal 3 IL) of OFQ-R are key amino acids to activateintracellular signal transduction via Gi causing inhibition of cAMPproduction.

Example 6 Amino Acids that Delineate Orphanin FQ and Dynorphin

Orphanin FQ is a heptadecapeptide with striking structural homology toDynorphin A (Dyn A), yet these peptides evoke opposite biologicalactions on colonic transit. This example describes the amino acidsequence delineation of Orphanin FQ and Dyn A. Infusion of OrphaninFQ(1–17) (100 nmol/kg/min) induced giant migrating contractions, whileDyn A (100 nmol/kg/min/) evoked non-migrating simultaneous contractions.Subcutaneous administration of Orphanin FQ (1–3 nmol/kg) acceleratedcolonic transit in a dose-dependent manner (geometric center increasedfrom 5.11±0.12 to 6.89±0.14 and 7.13±0.21, respectively). In contrast,subcutaneous administration of Dyn A (30 and 100 nmol/kg) yieldedsignificantly lower geometric center values: 3.14±0.21 and 2.42±0.17.Orphanin FQ(1–13) had the same potency as full peptide OrphaninFQ(1–17). Orphanin FQ(1–12) was 100 times less potent and OrphaninFQ(1–11) did not stimulate contractions. Differences were found in thestructural requirements for Orphanin FQ activity compared to Dyn A.Position 1 in the Orphanin FQ primary structure did not stringentlyrequire a Phe residue; substitution by tyrosine caused no loss ofactivity. Replacement by D-Phe produced partial activity, but an alaninesubstituted analogue was inactive. Thus, it appears that an aromaticside chain in position 1 is a minimal requirement for Orphanin activity.In contrast, any change of the Tyr position in Dyn A led to completeloss of activity. In Orphanin FQ, any change of the Phe residue inposition 4 or replacement of Arg⁸ by L-alanin resulted in complete lossof activity while a conformational conversion in position 8 had muchless effect. Thus, the positive charge of the arginine side chain and toa lesser extent its proper orientation may be important for biologicalactivity. The charge may interact with the side chain of a Glu or Aspresidue on the receptor and serve as an anchor. Studies indicate thatthe Orphanin FQ molecule contains a domain between amino acids 10 and 15that excludes it from activating the opiate receptor. Dyn A alsocontains an Orphanin FQ receptor “excluding” the region between aminoacids 7 and 10 together with Asp.

Example 7 Reversal of Postoperative Ileus by Orphanin Fq

Postoperative ileus (PI) often occurs after abdominal surgery. Thoughmany factors have been proposed, the pathophysiology of PI remainsunclear. The present invention is not limited to a particular mechanism.Indeed, an understanding of the mechanism is not necessary to practicethe present invention. Nonetheless, research suggests that thepurinergic pathway plays an important role in inhibition of colonicmotility. Further, adenosine A₁ receptor blockade appears to reverseexperimental PI in rats.

In vitro and in vivo colonic motility studies were performed using aknown rat model of PI. Rats equipped with a colonic cannula underwent asham operation or laparotomy plus simple cecum manipulation. Circularmuscle strips of distal colon were studied in vitro 4 h after surgery.Cecum manipulation reduced spontaneous colonic muscle contraction by 50%compared to sham operation. Suramin (nonselective P₂ purinoceptorantagonist, 10⁻⁴ mol/L) and reactive blue 2 (P_(2Y) purinoceptorantagonist, 3×10⁻⁵ mol/L), but not PPADGS (P_(2X) purinoceptorantagonist, 3×10⁻⁵ mol/L), greatly enhanced spontaneous contraction incircular muscle strips from rats with PI (210±15%, 287±20%, and 5±4%over basal, respectively). Similarly, OFQ (10⁻⁷ mol/L) reversed theinhibitory effects; contraction increased 450±60% over basal. Study ofthe purinergic pathway showed that cecum manipulation increased colonicP_(2Y1) and P_(2Y2) receptor mRNA expression by 38% and 63% compared tosham operation. Immunostaining revealed parallel findings. ATP inmyenteric neurons increased 2.5-fold, as measured by quinacrineimmunofluorescence. In response to electrical field stimulation, ATPrelease (luciferase assay) from colonic tissue after cecum manipulationwas 50% more than after sham operation. In vivo measurement of colonictransit using carmine migration was performed 4 h after cecummanipulation. In sham-operated rats, carmine migrated 90.8±6.5% of thecolon length in 2 h. Cecum manipulation slowed transit 68±5% compared tosham operation. OFQ dose-dependently reversed delayed colonic transitevoked by cecum manipulation (0.5 and 1 nmol/L OFQ improved transit to76±4% and 94±3%). In conclusion, the results presented hereindemonstrate that P_(2Y) purinoceptors on smooth muscle and ATPproduction in myenteric neurons increase in PI. These changes contributeto delayed colonic transit. OFQ, a potent inhibitor of purinergictransmission, reversed PI in rats.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in relevantfields are intended to be within the scope of the following claims.

1. A method of treating a subject having a colonic transit disorder,comprising: administering to said subject an effective amount of atherapeutic agent wherein said therapeutic agent binds to an Orphanin FQreceptor and said colonic transit disorder is ameliorated, and whereinsaid therapeutic agent is a polypeptide comprising a variant form of SEQID NO: 24 having at least one point mutation selected from the groupconsisting of R8K and K13C, wherein said variant form of SEQ ID NO: 24binds said Orphanin FQ receptor.
 2. The method of claim 1, wherein saidsubject is a mammal.
 3. The method of claim 2, wherein said mammal is ahuman.
 4. The method of claim 1, wherein said colonic transit disorderis selected from the group consisting of encopresis, colonic inertia,and irritable bowel disorder.